OAR polynucleotides, polypeptides and their use in PHA production in plants

ABSTRACT

The invention relates to the genes encoding 3-oxoacyl-[acyl carrier protein(ACP)] reductase (OAR). Compositions and methods for producing polyhydroxyalkanoate in transformed plants and transformed host cells are provided. Such methods find use in producing biodegradable thermoplastics in host cells and plants. Isolated nucleotide molecules, expression cassettes, isolated polypeptides and genetically manipulated host cells, plants, plant tissues, plant cells and seeds are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/258,417, filed Dec. 27, 2000, which is herebyincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[0002] The invention relates to compositions and methods for producingpolyhydroxyalkanoates in transformed plants and transformed host cells.

BACKGROUND OF THE INVENTION

[0003] Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoateswhich are naturally produced by a large variety of bacteria and fungi.PHAs are biodegradable and renewable, thereby providing an attractivealternative to petroleum-based plastics. However, high production costhas limited the widespread use of PHAs derived from bacterialfermentation. One alternative to reduce cost, production of PHAs inagricultural crops, has been regarded as promising. Small amounts of thePHA have been produced in the cytosol, plastids and peroxisomes ofgenetically engineered plants. See Poirier (1999) Curr. Opin.Biotechnol. 10(2):181-5; Madison et al. (1999) Microbiol. Mol. Biol Rev.63(1):21-53).

[0004] PHA synthases catalyze polymerization of hydroxyacyl-CoAsubstrates into PHA. The substrate specificity of this class of enzymesvaries across the spectrum of PHA-producing organisms. The variation insubstrate specificity of PHA synthases is supported by indirect evidenceobserved in heterologous expression studies (Lee et al. (1995) Appl.Microbiol Biotechnol. 42:901 and Timm et al. (1990) Appl. Microbiol.Biotech. 33:296).

[0005] Until recently, the only PHA that has been produced in plants waspolyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyric acid (Johnet al. (1996) Proc. Natl Acad. Sci. USA 93:12768-12773; Nawrath et al(1994) Proc. Natl. Acad. Sci. USA 91:12760-12764; Padgette et al. (1997)Plant Physiol. 114 (Suppl.) 3S; Poirier et al. (1992) Science256:520-523)). Because this polymer is crystalline and brittle with amelting point too close to its degradation point, PHB is difficult tomold into desirable products (Lee (1996) Biotechnol. Bioeng. 491:1-14).

[0006] Many bacteria make copolymers of 3-hydroxyalkanoic acids with acarbon chain length greater than or equal to five (Steinbuchel (1991)Biomaterials: Novel Materials from Biological Materials, ed. Byrom (NewYork: Macmillan Publishers Ltd.), pp. 123-213). Such copolymers arepolyesters composed of different 3-hydroxyalkanoic acid monomers.Depending on the composition, these copolymers can have propertiesranging from firm to elastic (Anderson et al. (1990) Microbiol. Rev.54:450-472; Lee, (1996) Biotechnol. Bioeng. 49:1-14). Unlike thehomopolymeric PHB, the PHA copolymers are suitable for a variety ofapplications because these copolymers exhibit a wide range of physicalproperties.

[0007] Initial attempts at producing PHA in plants involved producingPHA in the cytosol, but production of PHA in this cellular compartmentproved toxic to the plant (Poirier et al. (1992) Science 256:520-523).This problem was overcome by targeting the PHA-producing enzymes toplastids (Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA91:12760-12764). In either cellular compartment, however, only PHB wasaccumulated, not any of the copolymers. With both of these methods, thegenes from Ralstonia eutropha were used. The PHA synthase of thisbacterium can utilize only short chain (C₃-C₅) monomers (Steinbuchel(1991) Biomaterials: Novel Materials from Biological Materials, ed.Byrom (New York: Macmillan Publishers Ltd.), pp. 123-213). Later,copolymer production in Arabidopsis and canola was reported by Slater etal. (1999) Nature Biotechnology 17: 1011-1016.

[0008] The synthesis of PHA containing 3-hydroxyalkanoic acid monomersranging from six to sixteen carbons in Arabidopsis thaliana was reported(Mittendorf et al. (1998) Proc. Natl. Acad. Sci. USA 95:13397-13402). Toaccumulate PHA, the Arabidopsis plants were transformed with anucleotide sequence encoding PHA synthase from Pseudomonas aeruginosathat was modified for peroxisome targeting by the addition of anucleotide sequence encoding the C-terminal 34 amino acids of a Brassicanapus isocitrate lyase. In these plants, PHA was produced inglyoxysomes, leaf-type peroxisomes, and vacuoles. However, PHAproduction was very low in the Arabidopsis plants, suggesting thateither the introduced PHA synthase did not function properly in theintended organelle, or more likely that the necessary substrates for theintroduced PHA synthase were present at levels that were limiting forPHA synthesis. While this report demonstrated that PHA can be producedin peroxisomes of plants, the level of PHA produced in the plants wasfar below levels necessary for the commercial production of PHA inplants. Thus, methods and compositions directed to increasing the levelof substrate for PHA synthases are needed for production of PHA inplants.

[0009] There are two types of fatty acid synthase (FAS). In type I FAS,various enzyme activities are located on different domains of amultifunctional protein. In type II FAS, these enzyme activities arecatalyzed by individual polypeptides. 3-oxoacyl-[acyl carrierprotein(ACP)] reductase (OAR) is a component of the type II FAS. Thisenzyme reversibly reduces β-ketoacyl-ACP, the condensation product of anacetyl residue and a nascent acyl-ACP, to β-hydroxyacyl-ACP. In vitro,OAR also uses 3-ketoacyl-CoA as a substrate to catalyze formation of3-hydroxyacyl-CoA. This use of 3-ketoacyl-CoA is at a lower efficiencythan the use of β-ketoacyl-ACP as substrate (Shimakata et al. (1982)Arch. Biochem. Biophys. 218(1): 77-91).

[0010] NADPH-dependent OAR from Spinacia oleracea has been described tocatalyze the forward reaction of reducing β-ketoacyl-ACP, more thanseventeen times faster than the reverse dehydrogenation reaction, atneutral or acidic pH. This OAR has also been shown to use onlyD-3-hydroxybutyryl-ACP as a substrate but not the L-form counterpart.

[0011] NADH-dependent forms of OARs have been described from plantspecies such as castor bean and avocado (Shimakata et al. (1982) Arch.Biochem. Biophys. 218(1): 77-91; Caughey et al. (1982) Eur. J Biochem.123(3): 553-61). Taguchi et al. have shown that over-expression of abacterial NADPH-dependent OAR increases D-3-hydroxyacyl-CoA monomersupply for PHA synthase and leads to accumulation of PHAs in E. coli(Taguchi et al. (1999) Fems. Microbiol. Lett.176(1): 183-190).

[0012] Thus, methods and compositions directed to plant OARs are neededfor increasing the level of substrate for PHA synthases, and forproduction of PHA in plants.

SUMMARY OF THE INVENTION

[0013] Compositions and methods directed to producing PHA in host cellsand plants are provided, including PHA copolymers. The compositions aredirected to isolated nucleic acid molecules encoding 3-oxoacyl-[acylcarrier protein(ACP)] reductase (OAR) polypeptides. Expression cassettescomprising the nucleotide sequences encoding the OAR enzymes are alsoprovided.

[0014] For PHA production in host cells, such as bacteria, with one ormore endogenous PHA synthases, the methods involve geneticallymanipulating the host cell to produce one or more OAR enzymes. Themethods comprise stably integrating in the genome of a host cellnucleotide sequences encoding OAR enzymes.

[0015] For PHA production in plants, the methods involve geneticallymanipulating a plant to produce one or more OAR enzymes. If desired, theplants can also be transformed with nucleotide sequences encodingadditional enzymes that are necessary for, or favorably affect, thesynthesis of PHA in the plants. Such enzymes include, for example, oneor more PHA synthases. The OAR enzymes, and any other desired enzymes,can be targeted in the plant to the peroxisomes by operably linking aperoxisome-targeting sequence to a sequence encoding the enzyme. Themethods comprise stably integrating in the genome of a plant nucleotideconstructs comprising nucleotide sequences encoding OAR enzymes, PHAsynthases, and/or any other desired enzymes for PHA synthesis in a plantor part thereof.

[0016] Also provided are plants, plant tissues, plant cells, and seedsthereof, that are genetically manipulated to produce one or more OARenzymes. Further provided are plants, plant tissues, plant cells, andseeds thereof comprising stably integrated in their genomes a nucleotidesequence encoding an OAR and a nucleotide sequence encoding a PHAsynthase. Such plants, plant tissues, plant cells, and seeds canadditionally comprise stably integrated in their genomes one or moreadditional nucleotide sequences which encode enzymes that favorablyaffect PHA synthesis.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Compositions and methods for the production of biodegradablepolyesters in plants and other organisms are provided. In particular,nucleotide sequences for 3-oxoacyl-[acyl carrier protein (ACP)]reductase (OAR) genes are provided. More particularly, oar1 andoar2genes from maize, and oar1 and oar2 genes from soybean are provided(SEQ ID NOs: 1, 3, 5, and 7). The sequences can be used in combinationwith other sequences, including but not limited to PHA and PHBsynthases, to produce polyhydroxyalkanoates. These sequences can beprovided with peroxisome-targeting sequences for targeting to theperoxisomes. Also provided are polypeptides encoded by such nucleotidesequences (SEQ ID NOs: 2, 4, 6, and 8).

[0018] The present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NOs: 2, 4, 6, and 8. The invention encompassesisolated or substantially purified nucleic acid or protein compositions.An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acid(including protein encoding sequences) can be free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, preferably culture medium represents lessthan about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursorsor non-protein-of-interest chemicals.

[0019] Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes generally do not encode fragment proteins retaining biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 20 nucleotides, about 50 nucleotides, about 100 nucleotides,and up to the full-length nucleotide sequence encoding the proteins ofthe invention.

[0020] A fragment of an OAR nucleotide sequence that encodes abiologically active portion of an OAR protein of the invention willencode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, or 550 contiguous amino acids, or up to the total number of aminoacids present in a full-length OAR protein of the invention (forexample, 318, 312, 320, and 299 amino acid for SEQ ID NOs: 2, 4, 6, and8 respectively). Fragments of an OAR enzyme nucleotide sequence that areuseful as hybridization probes or PCR primers generally need not encodea biologically active portion of an OAR enzyme.

[0021] Thus, a fragment of an OAR enzyme nucleotide sequence may encodea biologically active portion of an OAR enzyme, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of an OAR enzyme can beprepared by isolating a portion of one of the OAR enzyme nucleotidesequences of the invention, expressing the encoded portion of the an OARenzyme (e.g., by recombinant expression in vitro), and assessing theactivity of the encoded portion of the a OAR enzyme. Nucleic acidmolecules that are fragments of an OAR enzyme nucleotide sequencecomprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300 350, 400, 450,500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, or up to thenumber of nucleotides present in a full-length OAR nucleotide sequencedisclosed herein (for example, 1326, 1286, 1398, and 1248 nucleotidesfor SEQ ID NOs: 1, 3, 5, and 7, respectively).

[0022] By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the OAR polypeptides of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode an OAR protein of theinvention. Generally, variants of a particular nucleotide sequence ofthe invention will have at least about 40%, 50%, 60%, 65%, 70%,generally at least about 75%, 80%, 85%, preferably at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97and more preferably at least about 98%,99% or more sequence identity to that particular nucleotide sequence asdetermined by sequence alignment programs described elsewhere hereinusing default parameters.

[0023] By “variant” protein is intended a protein derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variant proteins which areencompassed by the invention, and which are variants of a native OARprotein of the invention will have greater than 60%, about 70%,generally at least about 75%, 80%, 85%, preferably at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about98%, 99%, or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs describedelsewhere herein using default parameters. A biologically active variantof a protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

[0024] The proteins of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of OAR proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference. Inthis aspect of the present invention, conservative substitutions, suchas exchanging one amino acid with another having similar properties, maybe used.

[0025] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Generally, such variants will continue to possess the desired activity.Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and preferablywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444.

[0026] The deletions, insertions, and substitutions of the proteinsequences encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays. That is, theactivity can be evaluated by OAR activity assays. See, for example,Shimakata et al. (1982) Arch.Biochem. Biophys. 218(1): 77-91; hereinincorporated by reference.

[0027] Variant nucleotide sequences and proteins also encompasssequences and proteins derived from a mutagenic and recombinogenicprocedure such as DNA shuffling. With such a procedure, one or moredifferent OAR coding sequences can be manipulated to create a new OARenzyme possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the OAR gene ofthe invention and other known OAR genes to obtain a new gene coding fora protein with an improved property of interest, such as an increasedK_(m) or co-substrate specificity in the case of an enzyme. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0028] The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly otherbacteria. In this manner, methods such as PCR, hybridization, and thelike can be used to identify such sequences based on their sequencehomology to the sequences set forth herein. Sequences isolated based ontheir sequence identity to the entire OAR sequences set forth herein orto fragments thereof are encompassed by the present invention. Suchsequences include sequences that are orthologs of the disclosedsequences. By “orthologs” is intended genes derived from a commonancestral gene and which are found in different species as a result ofspeciation. Genes found in different species are considered orthologswhen their nucleotide sequences and/or their encoded protein sequencesshare substantial identity as defined elsewhere herein. Functions oforthologs are often highly conserved among species.

[0029] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); and Innis and Gelfand, eds.(1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand,eds. (1999) PCR Methods Manual (Academic Press, New York). Known methodsof PCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0030] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the OAR sequences of the invention. Methods for preparation of probesfor hybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0031] For example, the entire OAR sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding OAR sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among OAR sequences and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding OAR sequences from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

[0032] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0033] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1×to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. The duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

[0034] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0035] Thus, isolated sequences that encode for an OAR protein and whichhybridize under stringent conditions to the OAR sequences disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

[0036] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0037] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0038] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0039] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent sequence identity betweenany two sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0040] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J Mol. Biol215:403 are based on the algorithm of Karlin and Altschul (1990) supra.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding a protein of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to a protein orpolypeptide of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST (in BLAST 2.0) can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively,PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the defaultparameters of the respective programs (e.g., BLASTN for nucleotidesequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

[0041] Unless otherwise stated, sequence identity/similarity valuesprovided herein refer to the value obtained using GAP version 10 usingthe following parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

[0042] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0043] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0044] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0045] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0046] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0047] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C. lower than the T_(m), depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

[0048] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0049] The invention encompasses nucleotide constructs comprising thenucleic acids of the invention, and fragments and variants thereof; andmethods utilizing these constructs. The nucleotide constructs of thepresent invention are not limited to nucleotide constructs comprisingDNA. Those of ordinary skill in the art will recognize that nucleotideconstructs, particularly polynucleotides and oligonucleotides, comprisedof ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

[0050] Furthermore, it is recognized that the methods of the inventionmay employ a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a nucleotide construct iscomprised of a coding sequence for a protein or an RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ anucleotide construct that is not capable of directing, in a transformedplant, the expression of a protein or an RNA.

[0051] In addition, it is recognized that methods of the presentinvention do not depend on the incorporation of the entire nucleotideconstruct into the genome, only that the plant or cell thereof isaltered as a result of the introduction of the nucleotide construct intoa cell. In one embodiment of the invention, the genome may be alteredfollowing the introduction of the nucleotide construct into a cell. Forexample, the nucleotide construct, or any part thereof, may incorporateinto the genome of the plant. Alterations to the genome of the presentinvention include, but are not limited to, additions, deletions, andsubstitutions of nucleotides in the genome. While the methods of thepresent invention do not depend on additions, deletions, orsubstitutions of any particular number of nucleotides, it is recognizedthat such additions, deletions, or substitutions comprise at least onenucleotide.

[0052] The nucleotide constructs of the invention also encompassnucleotide constructs that may be employed in methods for altering ormutating a genomic nucleotide sequence in an organism, including, butnot limited to, chimeric vectors, chimeric mutational vectors, chimericrepair vectors, mixed-duplex oligonucleotides, self-complementarychimeric oligonucleotides, and recombinogenic oligonucleobases. Suchnucleotide constructs and methods of use, such as, for example,chimeraplasty, are known in the art. Chimeraplasty involves the use ofsuch nucleotide constructs to introduce site-specific changes into thesequence of genomic DNA within an organism. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

[0053] Methods are provided for producing PHA in host cells. Such hostcells find use in the production of biodegradable thermoplastics. Themethods involve transforming a host cell with a nucleic acid moleculeencoding an OAR enzyme, including but not limited to the nucleic acidmolecules of the invention encoding an OAR enzyme. Examples of known OARencoding nucleic acids that can be used in the methods of the inventioninclude those OAR encoding sequences described in Genbank accessionnumber AF042860, EMBL accession number X75781, EMBL accession numberX64566, and Genbank accession number T15143. While the invention is notlimited to any particular mechanism, it is envisioned that activeexpression of one or more OAR encoding sequences leads to reduction of3-ketoacyl-CoAs by the expressed OAR proteins, to form3-hydroxyacyl-CoAs. In turn, 3-hydroxyacyl-CoAs are used as substrate byone or more PHA synthases, for PHA synthesis. 3-ketoacyl-CoAs arenaturally produced in plants as intermediates in the β-oxidation cycle.

[0054] The methods additionally comprise growing the host cells for asufficient length of time in conditions favorable for the production ofPHA. The methods further involve extracting the PHA from the host cellsor from the vicinity of the host cells, such as for example, a culturebroth or solid medium. Host cells include non-human host cells includingbut not limited to plant cells, bacterial cells, yeast cells, fungalcells, algal cells and animal cells such as, for example, insect cellsand nematode cells. The host cells of the invention may be single cells,colonies or clumps of cells, or cells within a multicellular structureor organism.

[0055] Methods for producing PHB in the cytosol or plastids of plantsand for producing PHA in plant peroxisomes are known in the art.However, such methods do not achieve the synthesis of high levels of PHAin plants. The nucleotide sequences of the present invention find use inimproved methods for transforming plant host cells and producing PHA inplants, particularly in the cellular compartments therein such as thecytosol, plastids and peroxisomes, more particularly in the peroxisomes.

[0056] Depending on the particular host cell, in addition totransforming with one or more OAR-encoding nucleic acid molecules, themethods further involve transforming the host cell with at least oneadditional nucleic acid molecule encoding a PHA synthase. In vitro, OARprefers 3-ketoacyl-ACP to 3-ketoacyl-CoA as a substrate. However, whilethe invention is not limited by any particular mechanism, it isenvisioned that in the methods of the present invention, the additionalPHA synthase activity converts D-3-hydroxyacyl-CoA formed by OARcatalysis into PHAs, thus driving the OAR utilization of 3-ketoacyl-CoAsubstrate to proceed at a much higher rate in vivo.

[0057] In one embodiment, the PHA synthase utilized in the methods ofthe invention catalyzes the synthesis of PHA copolymers. By “PHAcopolymer” is intended a polymer composed of at least two different3-hydroxyalkanoic acid monomers. In another embodiment, such a PHAsynthase catalyzes the synthesis of PHA copolymers comprised of3-hydroxybutyric acid momomers and at least one additional monomerhaving a chain length of greater than four carbons. In yet anotherembodiment, such a PHA synthase catalyzes the synthesis of copolymerscomprised of 3-hydroxybutyric acid monomers and at least one additionalmonomer having a hydroxyacyl-chain length of from about 5 to about 18carbons. In further embodiments of the invention, the majority of PHAcopolymers produced are comprised of monomers of chain-length C₄ to C₁₈.

[0058] PHA synthases utilized in the methods of the invention includethose encoded by nucleotide sequences isolatable from Pseudomonasoleovorans (GenBank Accession No. M58445), Pseudomonas putida (AccessionNo. GenBank AF042276), Pseudomonas aeruginosa (EMBL Accession No.X66592), Aeromonas caviae (DDBJ Accession No. D88825) and Thiocapsapfennigii (EMBL Accession No. A49465). Such PHA synthases additionallyinclude the PHA synthases encoded by nucleotide sequences isolated fromPseudomonas fluorescens disclosed in WO 01/23580.

[0059] PHA synthases utilized in the methods of the invention arespecific to the R- (or D-) forms of their substrate 3-hydroxyacyl-CoAs.Consistent with this specificity, the OAR enzymes of the presentinvention catalyze the formation of R- (or D-) 3-hydroxyacyl-CoAs. Forthe purposes of the present invention, R-3-hydroxyacyl-CoAs andD-3-hydroxyacyl-CoAs are used interchangeably.

[0060] Methods for producing PHA in plants are provided. The methodsinvolve genetically manipulating the genome of a plant to produce PHA.The invention encompasses plants and seeds thereof, that have beengenetically manipulated to produce enzymes leading to PHA synthesis andexpression cassettes containing coding sequences for such enzymes. Theinvention further encompasses genetically manipulated plant cells andplant tissues. More particularly, the invention encompasses plants, andseeds thereof, that have been genetically manipulated to produce OARenzymes that produce substrates for PHA synthases, as well as thoseplants additionally manipulated to produce PHA synthases for utilizingthese substrates.

[0061] The methods provided for producing PHA involve geneticallymanipulating the plant to produce at least one OAR enzyme to catalyzeformation of substrate for PHA synthases. The plants of the inventioneach comprise in their genomes at least one stably incorporatednucleotide construct, each nucleotide construct comprising a codingsequence for an OAR enzyme operably linked to a promoter that drives theexpression of a gene in a plant. Plants of the invention are geneticallymanipulated to produce an OAR enzyme of the invention. In oneembodiment, the plants of the invention are additionally geneticallymanipulated to produce one or more PHA synthases, including, but notlimited to, the PHA synthases described above. Such PHA synthasesinclude those that are known to catalyze the synthesis of PHAcopolymers. Such PHA synthases include, but are not limited to, thosedescribed in WO 01/23596; and those disclosed in WO 01/23580 (providedas SEQ ID NOs:9 and 10 in the sequence listing for the presentapplication).

[0062] Additionally, a plant of the invention may comprise in its genomea nucleotide construct comprising a coding sequence for a PHA synthasecapable of synthesizing PHB including, but not limited to, those encodedby nucleotide sequences isolated from Ralstonia eutropha (GenBankAccession No. J05003), Acinetobacter sp. (GenBank Accession No. U04848),Ralstonia latus (GenBank Accession No. AF078795), Azorhizobiumcaulinodans (EMBL Accession No. AJ006237), Comamonas acidovorans (DDBJAccession No. AB009237), Methylobacterium extorquens (GenBank AccessionNo. L07893), Paracoccus denitrificans (DDBJ Accession No. D43764) andZoogloea ramigera (GenBank U66242). Thus, the invention encompassesplants, and seeds thereof, that have been genetically manipulated toproduce enzymes that produce substrates for PHB synthases; as well asthose additionally manipulated to produce PHB synthases for utilizingthe substrates.

[0063] Any method for producing more than one enzyme in a plant may beutilized. In one embodiment, a plant is transformed with more than oneconstruct in a transformation method. In another embodiment, a plant istransformed with one or more constructs comprising coding sequences formore than one enzyme described herein. In yet another embodiment, atransformed plant is re-transformed with one or more constructs. In afurther embodiment, transformed plants are crossed with one another, toproduce a plant producing more than one enzyme.

[0064] The nucleotide constructs of the invention each comprise a codingsequence for an OAR enzyme operably linked to a promoter that drivesexpression in a plant cell. Preferably, the promoters are selected fromseed-preferred promoters, chemical-regulatable promoters,germination-preferred promoters and leaf-preferred promoters. In anembodiment of the present invention, each of the nucleotide constructsadditionally comprises an operably linked nucleotide sequence encoding aperoxisome-targeting signal. The peroxisome-targeting signal may benative or endogenous to the particular OAR sequence, or it may be aheterologous peroxisome-targeting signal. It is recognized that, whereplants are genetically manipulated to produce one or more OAR enzymesand one or more PHA synthases and where expression of the OAR codingsequences in the peroxisome is desired, each nucleotide constructcomprising the OAR or PHA synthase coding sequence also comprises anoperably linked peroxisome-targeting signal.

[0065] OAR enzymes of the invention include those that prefer NADH toNADPH as co-substrate since NADH is the predominant electron donor inthe peroxisome. OAR enzymes of the invention also include those havingkinetic properties that strongly favor the forward reaction, reductionof 3-ketoacyl-CoAs, and disfavor the reverse reaction, thedehydrogenation of D-3-hydroxyacyl-CoAs. The OAR enzymes of theinvention further include, but are not limited to, those that are activein peroxisomal matrices. Additionally, the OAR enzymes or polypeptidesof the invention include, but are not limited to, those that catalyzethe formation of 3-hydroxyacyl-ACP, 3-hydroxyacyl-CoA, or both3-hydroxyacyl-ACP and 3-hydroxyacyl-CoA.

[0066] The OAR enzyme sequences of the invention can possess one or moreof the properties described above, including the ability to be targetedto peroxisomes, the ability to utilize NADH as co-substrate, thepreference for the forward reaction of reduction of 3-ketoacyl-CoAs, andthe ability to be active in the acidic environment of peroxisomes.Alternatively, these characteristics can be introduced into an OARsequence of the invention by methods known in the art and otherwisedescribed herein, such as site directed mutagenesis and DNA shuffling.For example, the NADH- or NADPH-binding domain of many proteins havebeen well characterized and a change of as few as three amino acids hasbeen shown to alter the binding specificity from NADPH to NADH(Nishiyama et al. (1993) J. Biol. Chem. 268(7): 465MO). Furthermore,NADH-preferring forms of OARs have been described from plant speciessuch as castor bean and avocado (Shimakata et al. (1982) Arch.Biochem.Biophys. 218(1): 77-91; Caughey et al. (1982) Eur. J. Biochem. 123(3):553-61), and can be used in DNA shuffling methods to conferNADH-preference to the OAR enzymes of the invention.

[0067] Alternatively, for utilizing NADPH-preferring OAR enzymes, it maybe necessary to increase the level of NADPH in the peroxisome. Thus, themethods of the invention may additionally involve stably integratinginto the genome of a plant a nucleotide construct comprising anucleotide sequence encoding an NADH kinase or an NAD kinase and anoperably linked promoter that drives expression in a plant cell. SuchNADH and NAD kinases catalyze the synthesis of NADPH and NADP⁺ ,respectively. Nucleotide sequences encoding such kinases include, butare not limited to DDJB Accession No. E131102 and EMBL Accession Nos.Z73544 and X84260. This construct may additionally comprise an operablylinked peroxisome-targeting signal sequence. By targeting such NADH andNAD kinases to the peroxisome, the level of NADPH and NADP⁺ can beincreased in the plant peroxisome for use by enzymes, such as, forexample, an NADPH-dependent 3-ketoacyl-CoA reductase.

[0068] Because PHA is not known to occur naturally in a plant, thebiosynthetic pathway for PHA in plant additionally encompasses enzymesand products thereof that are involved in PHA synthesis which resultfrom the genetic manipulation of the plant. By “intermediate molecule”is intended a precursor in the biosynthetic pathway for PHA in a plant.Intermediate molecules of the present invention include, but are notlimited to, fatty acids and β-oxidation products derived therefrom,acetyl-CoA, acetoacetyl-CoA and other 3-ketoacyl-CoAs,3-hydroxybutyryl-CoA and other 3-hydroxyacyl-CoAs.

[0069] Thus, it is recognized that for producing high levels of PHAcopolymers in certain plants, particularly in their peroxisomes, it maybe necessary to genetically manipulate plants to produce other enzymesinvolved in PHA synthesis; in addition to the OAR enzymes of theinvention. Generally, these other enzymes are directed to the peroxisometo increase the synthesis of at least one intermediate molecule leadingto increased levels of 3-ketoacyl-CoA

[0070] For example, such an intermediate molecule may be a precursor for3-ketoacyl-CoA synthesis; including but not limited to3-hydroxyacyl-CoA, enoyl-CoA, and acyl-CoA; the formations of which arecatalyzed by enoyl-CoA hydratase, acyl-CoA oxidase, and acyl-CoAsynthetase, respectively. Thus, it is recognized that depending on theparticular plant, endogenous levels of the OAR substrate,3-ketoacyl-CoA, may be limiting the level of D-3-hydroxyacyl-CoA thatcan be produced by the expressed OAR. Alternatively, or additionally, itis recognized that levels of 3-ketoacyl-CoA for utilization by theexpressed OAR can be increased by using antisense constructions todecrease or eliminate downstream utilization of 3-ketoacyl-CoA byenzymes other than OAR. For example, antisense constructs to the enzymethiolase can be used to increase the levels of 3-ketoacyl-CoA to beutilized by the expressed OAR. It is recognized that, where increasedproduction of D-(−)-3-hydroxybutyryl-CoA is desired, decrease orelimination of ketothiolase would not be desired.

[0071] Antisense constructions complementary to at least a portion ofthe messenger RNA (mRNA) for a corresponding sequence encoding an enzymecan be constructed. Antisense nucleotides are constructed to hybridizewith the corresponding mRNA. Modifications of the antisense sequencesmay be made as long as the sequences hybridize to and interfere withexpression of the corresponding mRNA. In this manner, antisenseconstructions having 70%, preferably 80%, more preferably 85% sequenceidentity to the corresponding antisensed sequences may be used.Furthermore, portions of the antisense nucleotides may be used todisrupt the expression of the target gene. Generally sequences of atleast 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater maybe used.

[0072] It is further recognized that levels of 3-ketoacyl-CoA forutilization by the expressed OAR can be increased by using constructionsin the sense orientation to decrease or eliminate downstream utilizationof 3-ketoacyl-CoA by enzymes other than OAR. That is, nucleotidesequences encoding endogenous enzymes catalyzing downstream utilizationof 3-ketoacyl-CoA may be used in the sense orientation to suppress theexpression of corresponding endogenous genes in plants. For example,sense constructs to the enzyme thiolase can be used to increase thelevels of 3-ketoacyl-CoA to be utilized by the expressed OAR. Methodsfor suppressing gene expression in plants using nucleotide sequences inthe sense orientation are known in the art. The methods generallyinvolve transforming plants with a nucleotide construct comprising apromoter that drives expression in a plant operably linked to at least aportion of a nucleotide sequence that corresponds to the transcript ofthe endogenous gene. Typically, such a nucleotide sequence hassubstantial sequence identity to the sequence of the transcript of theendogenous gene, preferably greater than about 65% sequence identity,more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

[0073] The methods of the invention comprise genetically modifyingplants to produce; in addition to the OAR and PHA synthase describedabove, one, two, three, four, five or more additional enzymes involvedin PHA synthesis. Examples of such enzymes include but are not limitedto enoyl-CoA hydratase, acyl-CoA oxidase, and acyl-CoA synthetase,Alternatively, or additionally, the methods of the invention comprisegenetically modifying plants to produce; in addition to the OAR and PHAsynthase described above, one, two, three, four, five or more antisenseconstructs corresponding to enzymes involved in PHA synthesis. In onemethod of the invention, each nucleotide construct comprising the codingsequence of one of these additional enzymes is operably linked to apromoter that drives expression in a plant and also to a nucleotidesequence encoding a peroxisome-targeting signal sequence. Depending onthe plant, the addition of one or more of these enzymes, and/orantisense constructs, may be necessary to achieve high-level PHAsynthesis in the plant.

[0074] The methods of the invention additionally comprise growing theplant under conditions favorable for PHA production, harvesting theplant, or one or more parts thereof, and isolating the PHA from theplant or part thereof. Such parts include, but are not limited to,seeds, leaves, stems, roots, fruits and tubers. The PHA may be isolatedor extracted from the plant or part thereof by methods known in the art.See, U.S. Pat. Nos. 5,942,597, 5,918,747, 5,899,339, 5,849,854 and5,821,299; herein incorporated by reference. See also, EP 859858A1, WO97/07229, WO 97/07230 and WO 97/15681; herein incorporated by reference.

[0075] The nucleotide sequences of the invention can be used in methodsfor producing PHA in plants. Such methods can be used in conjunctionwith methods known in the art for producing PHA in plants, particularlyin peroxisomes. The methods of the invention encompass utilizing thenucleotide sequences of the invention to increase the synthesis of anintermediate molecule in PHA synthesis. Such an intermediate moleculecan be limiting for PHA synthesis in the peroxisome and increasing thesynthesis of such a molecule in the peroxisome increases the level ofPHA produced in a plant. Intermediate molecules that can be limiting forPHA synthesis include, for example, R-(−) -3-hydroxybutyryl-CoA, otherR-(−)-3-hydroxyacyl-CoAs, acetoacetyl-CoA and other 3-ketoacyl-CoAs. Itis recognized that increasing the synthesis of an intermediate moleculein a plant peroxisome may not lead to an increased level of theintermediate molecule in the plant because the intermediate molecule maybe further metabolized into, for example, PHA.

[0076] The nucleotide sequences of the invention can be used inconjunction with other methods for producing PHA in plants including,but not limited to, methods that involve utilizing 3-ketoacyl-CoAreductase to form 3-hydroxyacyl-CoA substrate for PHA synthases, and/ormethods that involve increasing the levels ofR-(−)-3-hydroxybutyryl-CoA, other R-(−)-3-hydroxyacyl-CoAs,acetoacetyl-CoA and other 3-ketoacyl-CoAs. Examples of such methods aredescribed in WO 01/23596 and WO 01/23580; both of which are herebyincorporated herein in their entirety by reference.

[0077] In this aspect, the invention provides methods for producingincreased levels of PHA in the peroxisomes of plants that involveincreasing the synthesis of one or more intermediate molecules in theperoxisome, including, and in addition to, increasing the level ofR-(−)-3-hydroxyacyl-CoAs by transformation with OAR-encoding sequences.In one aspect of the invention, plants are genetically manipulated toincrease the synthesis of R-(−)-3-hydroxyacyl-CoAs. In a second aspectof the invention, plants are genetically manipulated to increase thesynthesis of a specific R-(−)-3-hydroxyacyl-CoA, R-(−)-3-hydroxybutyryl-CoA. In a third aspect of the invention, the first andsecond aspects are combined to provide plants that are geneticallymanipulated to increase the synthesis of both R-(−)-3-hydroxyacyl-CoAsand R-(−)-3-hydroxybutyryl-CoA.

[0078] Further, it is recognized that each of the aspects of theinvention may be used to produce PHA with substantially differentmonomer compositions. In particular, the level of 3-hydroxybutyric acidin the PHA produced in a plant will vary with each aspect. For eachparticular type of plant, PHA produced by plants of the second or thirdaspect of the invention is expected to have a higher 3-hydroxybutyricacid monomer content than PHA produced by plants of the first aspect.Similarly, PHA produced by plants of the second aspect is expected tohave a higher 3-hydroxybutyric acid monomer content than PHA produced byplants of the third aspect.

[0079] In a first embodiment of the invention, methods are provided forproducing PHA involving genetically manipulating a plant for increasedsynthesis of R-(−)-3-hydroxyacyl-CoA, a key intermediate molecule in PHAsynthesis in the peroxisome. The methods comprise stably integratinginto the genome of a plant one or more nucleotide constructs comprisinga coding sequence for an enzyme that catalyzes the formation ofR-(−)-3-hydroxyacyl-CoA substrate of PHA synthase, wherein at least onesuch construct comprises at least one OAR nucleotide sequence describedherein. Additionally, the plant can also comprise one or more nucleotideconstructs comprising a coding sequence for a PHA synthase. Inβ-oxidation in plant peroxisomes, acyl-CoA oxidase catalyzes theconversion of fatty acyl-CoA into 2-enoyl-CoA which is subsequentlyconverted to S-(+)-3-hydroxyacyl-CoA via the 2-enoyl-CoA hydratase of amultifunctional protein. While some R-(−)-3-hydroxyacyl-CoA may bepresent in peroxisomes, the level is believed to be very low andinsufficient to allow for the synthesis of an economically acceptablelevel of PHA in a plant. Furthermore, all known PHA synthases requirethat 3-hydroxyacyl-CoA monomers to be in R-(−)-form for PHA synthesis.To overcome the substrate limitation for PHA synthesis, the presentinvention discloses methods for PHA synthesis which involve providing aplant with an OAR enzyme in its peroxisomes that catalyzes the formationof R(−)-3-hydroxyacyl-CoA. By genetically manipulating a plant toincrease the synthesis of R-(−)-3-hydroxyacyl-CoA, the present inventionovercomes a major impediment to achieving high-level production of PHAcopolymers in plants.

[0080] In addition to an OAR enzyme described herein, the presentinvention encompasses utilizing an enoyl-CoA hydratase that catalyzesthe synthesis of R-(−)-3-hydroxyacyl-CoA, particularly a 2-enoyl-CoAhydratase from Aeromonas caviae. Alternatively, two proteins from yeastmay each be utilized as the additional enzyme. One such protein is theyeast multifunctional protein (GenBank Accession No. M86456) whichpossesses a 2-enoyl-CoA hydratase activity and a 3-hydroxyacyl-CoAdehydrogenase activity. The hydratase activity of the multifunctionalprotein is known to yield R-(−)-3-hydroxyacyl-CoA products. Ifnecessary, the dehydrogenase activity may be neutralized by methodsknown to those of ordinary skill in the art such as, for example,site-directed mutagenesis, and truncation of the coding sequence to onlythe portion necessary to encode the desired hydratase activity. Theother yeast protein is an enzyme identified as a 3-hydroxybutyryl-CoAdehydrogenase (Leaf et al. (1996) Microbiology 142:1169-1180). The geneencoding this enzyme may be cloned from Saccharomyces cerevisiae,sequenced and employed in the methods of the present invention. It isrecognized that the nucleotide sequence encoding this enzyme may need tobe modified to alter the amino acid sequence of the enzyme in such amanner as to favorably affect the production of R-(−)-3-hydroxyacyl-CoAin a plant. Such modifications may affect characteristics of the enzymesuch as, for example, substrate specificity, product specificity,product inhibition substrate binding affinity, product binding affinity,and the like. A method such as, for example, DNA shuffling may beemployed to modify this enzyme in the desired manner. Any method knownin the art for altering the characteristics of an enzyme to favorablyaffect the mass action ratio toward the desired product is encompassedby the methods of the present invention. Such methods typically involvemodifying at least a portion of the nucleotide sequence encoding theenzyme and include, but are not limited to, DNA shuffling, site-directedmutagenesis, and random mutagenesis.

[0081] In a second embodiment of the invention, methods for producingPHA are provided which involve genetically manipulating a plant forincreased synthesis of R-(−)-3-hydroxybutyryl-CoA, a substrate of PHAsynthase, in peroxisomes. The methods of the invention provide a plantthat is genetically manipulated for increased synthesis of a substratefor a PHA synthase and thus provide a plant that is geneticallymanipulated for high-level PHA synthesis in its peroxisomes. The methodsinvolve stably integrating into the genome of a plant one or moreprimary nucleotide constructs comprising a coding sequence for a3-ketoacyl-CoA reductase, wherein at least one such construct comprisesa nucleotide sequence encoding an OAR enzyme of the invention. Inanother embodiment the methods further involve stably integrating intothe genome of a plant one or more secondary nucleotide constructscomprising a coding sequence for a PHA synthase. In another embodiment,the methods further involve stably integrating into the genome of aplant one or more tertiary nucleotide constructs comprising a codingsequence for an acetyl-CoA:acetyl transferase. The primary, secondary,and tertiary constructs each additionally comprise an operably linkedpromoter that drives expression in a plant cell, and if necessary, anoperably linked peroxisome-targeting signal sequence. Acetyl-CoA:acetyltransferase, also referred to as ketothiolase, catalyzes the synthesisof acetoacetyl-CoA from two molecules of acetyl-CoA. Acetoacetyl-CoA maythen be converted into R-(−)-3-hydroxybutyryl-CoA via a reactioncatalyzed by an enzyme having 3-ketoacyl-CoA reductase activity, such asan OAR enzyme of the invention. The PHA synthase utilized in thisembodiment of the invention includes a PHB synthase and/or any PHAsynthase capable of accepting C₄ substrate, including PHA synthases thataccept C₄- and longer substrates.

[0082] 3-ketoacyl-CoA reductases utilized in the methods of theinvention are those that utilize NADH and include, but are not limitedto, at least a portion of the multifunctional proteins from yeast(GenBank Accession No. M86456), and rat (GenBank Accession No. U37486),wherein such a portion comprises a 3-ketoacyl-CoA reductase domain.

[0083] In the methods of the invention, however, NADPH-dependent3-ketoacyl-CoA reductases can also be employed including, but notlimited to, the 3-ketoacyl-CoA reductases encoded by GenBank AccessionNo. J04987 and EMBL Accession No. Z80156. Acetyl-CoA:acetyl transferasesthat can be utilized in the methods of the invention include, but arenot limited to a radish acetyl-CoA:acetyl transferase encoded by thenucleotide sequence having EMBL Accession No. X78116.

[0084] If necessary to increase the level of NADPH in the peroxisome,the methods of this embodiment may additionally involve, stablyintegrating into the genome of a plant, a quaternary nucleotideconstruct comprising a nucleotide sequence encoding a NADH kinase or anNAD kinase and an operably linked promoter that drives expression in aplant cell. Such NADH and NAD kinases catalyze the synthesis of NADPHand NADP⁺, respectively. Nucleotide sequences encoding such kinasesinclude, but are not limited to, DDJB Accession No. E131102 and EMBLAccession Nos. Z73544 and X84260. The fourth construct may additionallycomprise an operably linked peroxisome-targeting signal sequence. Bytargeting such NADH and NAD kinases to the peroxisome, the level ofNADPH and NADP⁺can be increased in the plant peroxisome for use byenzymes, such as, for example, an NADPH-dependent 3-ketoacyl-CoAreductase.

[0085] In a third embodiment of the invention, methods are provided forproducing PHA in a plant involving genetically manipulating a plant forincreased synthesis of R-(−)-3-hydroxybutyryl-CoA and otherR-(−)-3-hydroxyacyl-CoA molecules. Such methods provide a plant that isgenetically manipulated to overcome substrate limitations for PHAcopolymer synthesis in its peroxisomes. The methods involve stablyintegrating into the genome of a plant one or more primary, secondary,tertiary and quaternary nucleotide constructs comprising codingsequences for an enzyme involved in PHA synthesis in a plant. Theprimary nucleotide construct comprises a coding sequence for an enzymethat catalyzes the synthesis of R-(−)-3-hydroxyacyl-CoA, wherein atleast one such construct comprises a nucleotide sequence encoding an OARenzyme of the invention. The secondary nucleotide construct comprises acoding sequence for a 3-ketoacyl-CoA reductase. The tertiary nucleotideconstruct comprises a coding sequence for a PHA synthase that is capableof catalyzing the synthesis of PHA copolymers. The quaternary nucleotideconstruct comprises a coding sequence for an acetyl-CoA:acetyltransferase. If desired, an additional nucleotide construct may also bestably integrated into the genome of the plant. The additionalnucleotide construct comprises a nucleotide sequence encoding a NADHkinase or an NAD kinase.

[0086] Nucleotide constructs that can be utilized in this thirdembodiment include the nucleotide constructs of the first and secondembodiments, described supra. The nucleotide constructs used in thisthird embodiment, each additionally comprises an operably linkedpromoter and, if necessary, an operably linked peroxisome-targetingsignal to direct the encoded protein to the peroxisome. By targetingsuch enzymes to the peroxisome, the plant is capable of increasedsynthesis of intermediate molecules, particularly intermediate moleculesthat are substrates for a PHA synthase that catalyzes the formation ofcopolymers.

[0087] It is recognized that the methods of the present invention can beused in combination with methods for producing PHA homopolymers,copolymers or both. Further, it is recognized that it may be necessaryto lower or eliminate the activity of an endogenous enzyme in a plantthat in some way limits the synthesis of the desired intermediatemolecule. Such an endogenous enzyme may, for example, catabolize ormodify the intermediate molecule in an undesirable way. Methods forlowering or eliminating the activity of an enzyme in a plant includesense and antisense suppression methods.

[0088] The OAR sequences of the invention are provided in expressioncassettes for expression in the plant of interest. The cassette willinclude 5′ and 3′ regulatory sequences operably linked to an OARsequence of the invention. By “operably linked” is intended a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame. In the case of protein coding sequences, “operablylinked” includes joining two protein coding sequences in such a mannerthat both sequences are in the same reading frame for translation. Forexample, a nucleotide sequence encoding a peroxisome-targeting signalmay be joined to the 3′ end of a coding sequence of a protein of theinvention in such manner that both sequences are in the same readingframe for translation to yield a the protein of the invention with aC-terminal addition of the peroxisome-targeting signal. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes.

[0089] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of an OAR sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

[0090] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of the invention, and a transcriptional and translationaltermination region functional in plants. The transcriptional initiationregion, the promoter, may be native or analogous or foreign orheterologous to the plant host. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. By “foreign” isintended that the transcriptional initiation region is not found in thenative plant into which the transcriptional initiation region isintroduced. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence.

[0091] The termination region may be native with the transcriptionalinitiation region, may be native with the operably linked DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0092] Where appropriate, the gene(s) may be optimized for increasedexpression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

[0093] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. When possible, the sequence is modified to avoid predicted hairpinsecondary mRNA structures.

[0094] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

[0095] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

[0096] A number of promoters can be used in the practice of theinvention. The promoters may be selected based on the desired timing,localization and level of expression genes encoding enzymes in a plant.Constitutive, seed-preferred, germination-preferred, tissue-preferredand chemical-regulatable promoters can be used in the practice of theinvention.

[0097] Such constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al (1985) Nature 313:810-812); rice actin (McElroy etal. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989)Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol.Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

[0098] The methods of the invention are useful for producing PHA inseeds. To drive the expression of an OAR nucleotide sequence of theinvention in seeds, seed preferred promoters can be operably linked toan OAR nucleotide sequence. “Seed-preferred” promoters include both“seed-specific” promoters (those promoters active during seeddevelopment such as promoters of seed storage proteins) as well as“seed-germinating” promoters (those promoters active during seedgermination). See Thompson et al. (1989) BioEssays 10:108, hereinincorporated by reference. Such seed-preferred promoters include, butare not limited to, Cim1 (cytokinin-induced message); cZ19B1(maize 19kDa zein); milps (myo-inositol-1-phosphate synthase); and celA(cellulose synthase) (see WO 00/11177, herein incorporated byreference). Gama-zein is a preferred endosperm-specific promoter. Glob-1is a preferred embryo-specific promoter. For dicots, seed-specificpromoters include, but are not limited to, bean β-phaseolin, napin,β-conglycinin, soybean lectin, cruciferin, and the like. For monocots,seed-specific promoters include, but are not limited to, maize 15 kDazein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2,globulin 1, etc. See also WO 00/12733, where seed-preferred promotersfrom end1 and end2 genes are disclosed; herein incorporated byreference.

[0099] For tissue-preferred expression, the coding sequences of theinvention can be operably linked to tissue-preferred promoters. Forexample, leaf-preferred promoters may be utilized if expression inleaves is desired. Leaf-preferred promoters are known in the art. See,for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773 -778; Gotor et al. (1993) Plant J. 3:509-18; Orozcoet al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al.(1993) Proc. Natl. Acad. Sci. USA 90(20):9586 -9590.

[0100] Other tissue-preferred promoters include, for example, Kawamataet al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997)Mol Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Lam (1994) Results Probl CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol Biol.23(6):1129-1138; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

[0101] In the practice of the invention, it may be desirable to usechemical-regulatable promoters to control the expression of gene in aplant. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulatable promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0102] In particular embodiments of the invention, the expressioncassette may additionally comprise a nucleotide sequence encoding aperoxisome-targeting signal, in order to direct an OAR to theperoxisomes of a plant. Methods for directing an enzyme to theperoxisome are well known in the art. Typically, such methods involveoperably linking a nucleotide sequence encoding a peroxisome-targetingsignal to the coding sequence of a protein or modifying the codingsequence to additionally encode the peroxisome-targeting signal withoutsubstantially affecting the intended function of the encoded protein.See, for example, Olsen et al (1993) Plant Cell 5:941-952, Mullen et al.( 1997) Plant Physiol. 115:881-889, Gould et al. (1990) EMBO J. 9:85-90,Flynn et al. (1998) Plant J. 16:709-720, Preisig-Muller and Kindl (1993)Plant Mol. Biol. 22:59-66, and Kato et al. (1996) Plant Cell8:1601-1611; herein incorporated by reference. In one embodiment, theperoxisome-targeting signal is a PTS 1-type peroxisomal targetingsignal.

[0103] It is recognized that an OAR of the invention may be directed tothe peroxisome by operably linking a peroxisome-targeting signal to theC-terminus or the N-terminus of the enzyme. It is further recognizedthat an enzyme which is synthesized with a peroxisome-targeting signalmay be processed proteolytically in vivo resulting in the removal of theperoxisome-targeting signal from the amino acid sequence of the mature,peroxisome-localized enzyme.

[0104] It is further recognized that the components of the expressioncassette may be modified to increase expression. For example, truncatedsequences, nucleotide substitutions or other modifications may beemployed. See, for example Perlak et al. (1991) Proc. Natl. Acad. Sci.USA 88:3324-3328; Murray et al. (1989) Nucleic Acid Research 17:477-498;and WO 91/16432.

[0105] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0106] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81 -84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.

[0107] In the methods of the present invention, plants geneticallymanipulated to produce PHA are utilized. By “genetically manipulated” isintended modifying the genome of an organism, preferably a plant,including cells and tissue thereof, by any means known to those skilledin the art. Modifications to a genome include both losses and additionsof genetic material as well as any sorts of rearrangements in theorganization of the genome. Such modifications can be accomplished by,for example, transforming a plant's genome with a nucleotide constructcontaining nucleotide sequences which are native to the recipient plant,non-native or a combination of both, conducting a directed sexual matingor cross pollination within a single species or between related species,fusing or transferring nuclei, inducing mutagenesis and the like.

[0108] In the practice of certain specific embodiments of the presentinvention, a plant is genetically manipulated to produce more than oneheterologous enzyme involved in PHA synthesis. Those of ordinary skillin the art realize that this can be accomplished in any one of a numberof ways. For example, each of the respective coding sequences for suchenzymes can be operably linked to a promoter and then joined together ina single continuous polynucleotide fragment comprising a multigenicexpression cassette. Such a multigenic expression cassette can be usedto transform a plant to produce the desired outcome. Alternatively,separate plants can be transformed with expression cassettes containingone or a subset of the desired set of coding sequences. Transformedplants that express the desired activity can be selected by standardmethods available in the art such as, for example, assaying enzymeactivities, immunoblotting using antibodies which bind to the enzymes ofinterest, assaying for the products of a reporter or marker gene, andthe like. Then, all of the desired coding sequences can be broughttogether into a single plant through one or more rounds of crosspollination utilizing the previously selected transformed plants asparents.

[0109] Methods for cross pollinating plants are well known to thoseskilled in the art, and are generally accomplished by allowing thepollen of one plant, the pollen donor, to pollinate a flower of a secondplant, the pollen recipient, and then allowing the fertilized eggs inthe pollinated flower to mature into seeds. Progeny containing theentire complement of heterologous coding sequences of the two parentalplants can be selected from all of the progeny by standard methodsavailable in the art as described supra for selecting transformedplants. If necessary, the selected progeny can be used as either thepollen donor or pollen recipient in a subsequent cross pollination.

[0110] The invention can be practiced with any plant. Plants of interestinclude, but are not limited to, corn (Zea mays), canola and otherBrassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus),cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocosnucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), oats, barley, vegetables,pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries),ornamentals, and conifers. In an embodiment of the invention, oilseedplants are transformed with the OAR nucleotide sequences of theinvention. Such oilseed plants include, but are not limited to canola,sunflower, safflower, soybean, peanut, cotton, flax, coconut and oilpalm.

[0111] Additionally, the OAR nucleotide sequences of the invention maybe used in methods for producing PHA in non-human host organisms otherthan plants; including but not limited to bacteria, yeasts, and fungi.Useful host organisms for PHA production include Actinomycetes (e.g.,Streptomyces sp. and Nocardia sp.); bacteria (e.g., Ralstonia (e.g., R.eutropha), Bacillus cereus, B. subtilis, B. licheniformis, B.megaterium, Escherichia coli, Klebsiella (e.g., K. aerogenes and K.oxytoca), Lactobacillus, Methylomonas, Pseudomonas (e.g., P. putida andP. fluorescens); fungi (e.g., Aspergillus, Cephalosporium, andPenicillium); and yeast (e.g., Saccharomyces, Rhodotorula, Candida,Hansenula, and Pichia).

[0112] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Isolation of Maize and Soybean OAR Genes

[0113] The dehydrogenase domain of rat multifunctional protein type 2(MFP2), were used as a query to initially identify maize OARs.Subsequently, two OAR cDNAs (ESTs) from maize (maize OAR1 and OAR2), andtwo OAR cDNAs (ESTs) from soybean (soybean OAR1 and OAR2) wereidentified. The corresponding cDNAs were isolated by known methods. Thenucleic acid sequences for these cDNAs, and the polypeptides encodedthereby, are set forth in SEQ ID Nos: 1-4 for the maize OARs, and SEQ IDNos: 5-8 for the soybean OARs. Sequence analysis indicates the presenceof putative N-terminal transit peptide for each gene. The two transitpeptides from the two soybean OARs are significantly different in lengthand amino acid composition, and may represent targeting signals fordifferent organelles.

[0114] The amino acid sequences for the maize and soybean polypeptideshave about 60% identity with an OAR sequence from Cuphea lanceolata(EMBL accession number X64566). They also share significant homologywith the dehydrogenase portion of mammalian and yeast D-specificperoxisomal multifunctional proteins (MFP2) that are involved in theβ-oxidation of fatty acids (Hashimoto, T. (1999) Neurochem. Res. 24(4):551-63).

Example 2 Production of Transgenic Dicotyledonous Soybean Plants viaBiolistic Transformation

[0115] For constitutive expression of the nucleic acids of theinvention, constructs comprising the SCP1 promoter (U.S. Pat. No.6,072,050) and the OAR coding regions of the invention are introducedinto embryogenic suspension cultures of soybean, or other dicots, byparticle bombardment using essentially the methods described in Parrottet al. (1989) Plant Cell Rep. 7: 615-617. For seed-preferred expression,constructs comprising the beta phaseolin promoter (van der Geest (1996)Plant Mol Biol. 32(4): 579-88; Slightom et al. (1983) Proc. Natl. AcadSci USA 80: 1897-1901) and the OAR coding regions of the invention areused for the particle bombardment.

[0116] Seed is removed from pods when the cotyledons are between 3 and 5mm in length. The seeds are sterilized in a Clorox bleach solution(0.5%) for 15 minutes after which time the seeds are rinsed with steriledistilled water. The immature cotyledons are excised by first cuttingaway the portion of the seed that contains the embryo axis. Thecotyledons are then removed from the seed coat by gently pushing thedistal end of the seed with the blunt end of the scalpel blade. Thecotyledons are then placed (flat side up) on SB1 initiation medium (MSsalts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH5.8). The Petri plates are incubated in the light (16 hour day; 75-80 μEat 26° C. After 4 weeks of incubation the cotyledons are transferred tofresh SB1 medium. After an additional two weeks, globular stage somaticembryos that exhibit proliferative areas are excised and transferred toFN Lite liquid medium (Samoylov et al. (1998) In Vitro Cell Dev.Biol.—Plant 34:8-13). About 10 to 12 small clusters of somatic embryosare placed in 250 ml flasks containing 35 ml of SB172 medium.

[0117] The embryogenic suspension cultures are maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights (20 μE on a 16:8 hour day/night schedule. Cultures aresub-cultured every two weeks by inoculating approximately 35 mg oftissue into 35 mL of liquid medium.

[0118] Embryogenic suspension cultures are then transformed usingparticle gun bombardment (Klein et al. (1987) Nature(London) 327:70,U.S. Pat. No. 4,945,050). A BioRad Biolistic™ PDS1000/HE instrument isused for these transformations. A selectable marker gene which can beused to facilitate transformation is a chimeric gene composed of the 35Spromoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225(from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region ofthe nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens.

[0119] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

[0120] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. Membrane rupture pressure is set at 1100psi and the chamber is evacuated to a vacuum of 28 inches mercury. Thetissue is placed approximately 8 cm away from the retaining screen, andis bombarded three times. Following bombardment, the tissue is dividedin half and placed back into 35 ml of FN Lite medium.

[0121] Five to seven days after bombardment, the liquid medium isexchanged with fresh medium. Eleven days post-bombardment the medium isexchanged with fresh medium containing 50 mg/mL hygromycin. Thisselective medium is refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue is observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue isremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each new lineis treated as an independent transformation event. These suspensions arethen subcultured and maintained as clusters of immature embryos, ortissue is regenerated into whole plants by maturation and germination ofindividual embryos. Tissue from the regenerated plant is tested for OARexpression using known methods for detecting gene expression; such asWestern and Northern Blotting. Active expression of the OAR protein istested by assaying for acetoacetyl-CoA- and/or acetoacetyl-ACP reductaseactivity. Such assay methods are known in the art. See, for example,Shimakata et al. (1982) Arch.Biochem. Biophys. 218(1): 77.

[0122] Alternatively, an Agrobacterium transformation method is used,for example, as described in Byrne et al. (1987) Plant Cell Tissue andOrgan Culture 8:3-15; Facciotti et al. (1985) Biotechnology (New York)3:241; or U.S. Pat. No. 5,569,834.

Example 3 Transformation and Regeneration of Transgenic Maize Plants byParticle Bombardment

[0123] For constitutive expression of an OAR nucleotide sequence of theinvention, immature maize embryos from greenhouse donor plants arebombarded with a plasmid containing a constitutive promoter operablylinked to an OAR nucleotide sequence of the invention and the selectablemarker gene PAT (Wohlleben et al. (1988) Gene 70:25-37), which confersresistance to the herbicide Bialaphos. For seed-preferred expression ofan OAR nucleotide sequence of the invention, immature maize embryos fromgreenhouse donor plants are bombarded with a plasmid containing aseed-preferred promoter operably linked to an OAR nucleotide sequence ofthe invention and the selectable marker gene PAT. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

[0124] Preparation of Target Tissue

[0125] The ears are husked and surface sterilized in 30% Clorox bleachplus 0.5% Micro detergent for 20 minutes, and rinsed two times withsterile water. The immature embryos are excised and placed embryo axisside down (scutellum side up), 25 embryos per plate, on 560Y medium for4 hours and then aligned within the 2.5-cm target zone in preparationfor bombardment.

[0126] Preparation of DNA

[0127] A plasmid vector comprising an OAR nucleotide sequence of theinvention operably linked to a constitutive or seed-preferred promoteris made. This plasmid DNA plus plasmid DNA containing a PAT selectablemarker is precipitated onto 1.1 μm (average diameter) tungsten pelletsusing a CaCl₂ precipitation procedure as follows:

[0128] 100 μl prepared tungsten particles in water

[0129] 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

[0130] 100 μl 2.5 M CaCl₂

[0131] 10 μl 0. 1 M spermidine

[0132] Each reagent is added sequentially to the tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

[0133] Particle Gun Treatment

[0134] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

[0135] Subsequent Treatment

[0136] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for OAR expression, for example.

[0137] Bombardment and Culture Media

[0138] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂ O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000XSIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

[0139] Plant regeneration medium (288J) comprises 4.3 g/l MS salts(GIBCO 11117 -074), 5.0 ml/l MS vitamins stock solution (0.100 gnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂O) (Murashige andSkoog (1962) Physiol. Plant. 15:473), 100 mg/lmyo-inositol, 0.5 mg/lzeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought tovolume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite(added after bringing to volume with D-I H₂O); and 1.0 mg/l indoleaceticacid and 3.0 mg/l bialaphos (added after sterilizing the medium andcooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MSsalts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/lnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂O), 0.1 g/lmyo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-IH₂O after adjusting pH to 5.6); and 6 g/l bacto-agar (added afterbringing to volume with polished D-I H₂O), sterilized and cooled to 60°C.

Example 4 Production of Transgenic Maize Plants viaAgrobacterium-Mediated Transformation

[0140] For Agrobacterium-mediated transformation of maize an OARnucleotide sequence of the invention, preferably the method of Zhao isemployed (U.S. Pat. No. 5,981,840, and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the OAR nucleotide sequence of the invention toat least one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are preferablyimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Preferably the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

Example 5 Production of PHA Copolymers in Plants

[0141] Transgenic plants expressing one or more OAR enzymes of theinvention are produced according to the methods illustrated in Examples2-4, or by any other method for producing transgenic plants that isknown in the art. Additionally, the plants are also transformed with,and express a PHA synthase, particularly a PHA synthase capable ofaccepting C₄-C₁₈ substrates. While such a PHA synthase will typically becapable of using monomers of 3-hydroxyalkanoic acids-CoAs withhydroxyalkanoate carbon chain lengths of C₄, C₅, C₆, C₇, C₈, C₉, C_(1O),C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆,C₁₇, and C₁₈, the invention does not dependon a particular PHA synthase being capable of utilizing all monomerswith the range of C₄-C₁₈. Furthermore, the invention encompasses the useof PHA synthases that do not utilize monomer all carbon chain lengthsfrom C₄-C₁₈. Alternatively, the plants are transformed with a PHBsynthase, as well as a PHA synthase capable of accepting substratelonger than C₄ PHA copolymer production is tested by methods known inthe art. For example, see Mittendorf et al. (1998) Proc. Natl. Acad.Sci. USA 95:13397-13402.

[0142] If desired, the plants can also be transformed as described suprawith nucleotide sequences encoding additional enzymes that are necessaryfor, or favorably affect, the synthesis of PHA in the plants. Suchenzymes include, for example, one or more PHA synthases. The OARenzymes, and any other desired enzymes, can be targeted in the plant tothe peroxisomes by operably linking a peroxisome-targeting sequence to asequence encoding the enzyme.

[0143] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0144] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 10 1 1326 DNA Zea mays CDS (157)...(1110) 1 gcgcggagct tccaaagcccccgtccccca atagactcct ccccatccgt gctctgctcc 60 gtcacggctc aaatactccgcctgcatctc caaagcacac tgctccctct ggcttcccgc 120 ctcctcttcg gctccttcgcgtcccgacgc cccctc atg gcc acc gcc gcc gcc 174 Met Ala Thr Ala Ala Ala 15 acc gca gca gca gca gca gtc tcc tcc ccg gct gcg cgt gga gca gcc 222Thr Ala Ala Ala Ala Ala Val Ser Ser Pro Ala Ala Arg Gly Ala Ala 10 15 20ggg gcc gcc gcc gcc tcc cgc cgg ggg ttc gtc acg ttt ggt gga ggc 270 GlyAla Ala Ala Ala Ser Arg Arg Gly Phe Val Thr Phe Gly Gly Gly 25 30 35 gccgcc cgc ttc tct ccc acg ctg cgg tcc ggc cgt ggg ttc tct ggt 318 Ala AlaArg Phe Ser Pro Thr Leu Arg Ser Gly Arg Gly Phe Ser Gly 40 45 50 gtg caaacc cat gtt gct gct gtt gaa caa gca gtt gta aaa gat gct 366 Val Gln ThrHis Val Ala Ala Val Glu Gln Ala Val Val Lys Asp Ala 55 60 65 70 acc aagctg gaa gct cca gtt gtt gtt gtt aca ggt gca tct aga ggg 414 Thr Lys LeuGlu Ala Pro Val Val Val Val Thr Gly Ala Ser Arg Gly 75 80 85 att ggt aaggca act gct cta gcc ctt gga aaa gca gga tgc aag gtt 462 Ile Gly Lys AlaThr Ala Leu Ala Leu Gly Lys Ala Gly Cys Lys Val 90 95 100 ctg gta aactat gcc cgg tcc tcg aaa gag gct gaa gag gtc tcc aaa 510 Leu Val Asn TyrAla Arg Ser Ser Lys Glu Ala Glu Glu Val Ser Lys 105 110 115 gag att gaagca tct ggt ggt gag gct atc acc ttc gga gga gat gtt 558 Glu Ile Glu AlaSer Gly Gly Glu Ala Ile Thr Phe Gly Gly Asp Val 120 125 130 tca aaa gaagct gat gta gag tct atg atg aaa gca gct cta gat aaa 606 Ser Lys Glu AlaAsp Val Glu Ser Met Met Lys Ala Ala Leu Asp Lys 135 140 145 150 tgg ggaaca ata gat gtg ctg gta aat aat gca ggg att aca cga gac 654 Trp Gly ThrIle Asp Val Leu Val Asn Asn Ala Gly Ile Thr Arg Asp 155 160 165 aca ttgttg atg agg atg aag aaa tct cag tgg caa gac gta att gat 702 Thr Leu LeuMet Arg Met Lys Lys Ser Gln Trp Gln Asp Val Ile Asp 170 175 180 ctg aatctt act ggc gtc ttc ctt tgt aca cag gct gca aca aaa gta 750 Leu Asn LeuThr Gly Val Phe Leu Cys Thr Gln Ala Ala Thr Lys Val 185 190 195 atg atgaaa aag aga aag gga aaa att atc aac att gca tct gta gtt 798 Met Met LysLys Arg Lys Gly Lys Ile Ile Asn Ile Ala Ser Val Val 200 205 210 ggt cttact ggc aat gtt ggc caa gct aat tat agc gca gcc aag gct 846 Gly Leu ThrGly Asn Val Gly Gln Ala Asn Tyr Ser Ala Ala Lys Ala 215 220 225 230 ggagtg att ggt ttc aca aaa aca gtt gcc agg gag tat gca agc aga 894 Gly ValIle Gly Phe Thr Lys Thr Val Ala Arg Glu Tyr Ala Ser Arg 235 240 245 aatatc aat gtg aat gct att gca cca ggg ttc att gca tct gat atg 942 Asn IleAsn Val Asn Ala Ile Ala Pro Gly Phe Ile Ala Ser Asp Met 250 255 260 actgcc gaa ctt gga gaa gag ctt gag aag aaa atc ttg tca acc att 990 Thr AlaGlu Leu Gly Glu Glu Leu Glu Lys Lys Ile Leu Ser Thr Ile 265 270 275 ccgtta ggg aga tat ggc caa cca gag gaa gtt gca ggg ttg gtc gag 1038 Pro LeuGly Arg Tyr Gly Gln Pro Glu Glu Val Ala Gly Leu Val Glu 280 285 290 ttcctg gcc ctt aac ccc gca gct agc tat atg act gga cag gtg ctt 1086 Phe LeuAla Leu Asn Pro Ala Ala Ser Tyr Met Thr Gly Gln Val Leu 295 300 305 310aca att gac gga ggg atg gta atg taagatttga gttagcttga tgcacttcta 1140Thr Ile Asp Gly Gly Met Val Met 315 cttttgctga gcatttaatg ttgaacacccttgttgtgca cgggcgattt tggacaacaa 1200 attagtgttg tctctttctt tgtaatactctctggtaata aatctagcat gtggaatgga 1260 agttgaaatc tgggttttcg tgtaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320 aaaaaa 1326 2 318 PRT Zea mays 2Met Ala Thr Ala Ala Ala Thr Ala Ala Ala Ala Ala Val Ser Ser Pro 1 5 1015 Ala Ala Arg Gly Ala Ala Gly Ala Ala Ala Ala Ser Arg Arg Gly Phe 20 2530 Val Thr Phe Gly Gly Gly Ala Ala Arg Phe Ser Pro Thr Leu Arg Ser 35 4045 Gly Arg Gly Phe Ser Gly Val Gln Thr His Val Ala Ala Val Glu Gln 50 5560 Ala Val Val Lys Asp Ala Thr Lys Leu Glu Ala Pro Val Val Val Val 65 7075 80 Thr Gly Ala Ser Arg Gly Ile Gly Lys Ala Thr Ala Leu Ala Leu Gly 8590 95 Lys Ala Gly Cys Lys Val Leu Val Asn Tyr Ala Arg Ser Ser Lys Glu100 105 110 Ala Glu Glu Val Ser Lys Glu Ile Glu Ala Ser Gly Gly Glu AlaIle 115 120 125 Thr Phe Gly Gly Asp Val Ser Lys Glu Ala Asp Val Glu SerMet Met 130 135 140 Lys Ala Ala Leu Asp Lys Trp Gly Thr Ile Asp Val LeuVal Asn Asn 145 150 155 160 Ala Gly Ile Thr Arg Asp Thr Leu Leu Met ArgMet Lys Lys Ser Gln 165 170 175 Trp Gln Asp Val Ile Asp Leu Asn Leu ThrGly Val Phe Leu Cys Thr 180 185 190 Gln Ala Ala Thr Lys Val Met Met LysLys Arg Lys Gly Lys Ile Ile 195 200 205 Asn Ile Ala Ser Val Val Gly LeuThr Gly Asn Val Gly Gln Ala Asn 210 215 220 Tyr Ser Ala Ala Lys Ala GlyVal Ile Gly Phe Thr Lys Thr Val Ala 225 230 235 240 Arg Glu Tyr Ala SerArg Asn Ile Asn Val Asn Ala Ile Ala Pro Gly 245 250 255 Phe Ile Ala SerAsp Met Thr Ala Glu Leu Gly Glu Glu Leu Glu Lys 260 265 270 Lys Ile LeuSer Thr Ile Pro Leu Gly Arg Tyr Gly Gln Pro Glu Glu 275 280 285 Val AlaGly Leu Val Glu Phe Leu Ala Leu Asn Pro Ala Ala Ser Tyr 290 295 300 MetThr Gly Gln Val Leu Thr Ile Asp Gly Gly Met Val Met 305 310 315 3 1286DNA Zea mays CDS (115)...(1050) 3 ccacaccaaa cgtgccaaac ccccaacgccatcctctata aacggcttcc tcgcgggctc 60 cccctccccc tccccgactc ctccccatcgcccatcgccg ccctccgatc cttc atg 117 Met 1 gcc gct gcc aca gcc gcc gcc gccgcg ctc gcc tcc ccg gcg ggc ctc 165 Ala Ala Ala Thr Ala Ala Ala Ala AlaLeu Ala Ser Pro Ala Gly Leu 5 10 15 tcc aca tcg ctg gcg cgc cgc ggc ctcgtc agc ttc gca ccc gcg ctc 213 Ser Thr Ser Leu Ala Arg Arg Gly Leu ValSer Phe Ala Pro Ala Leu 20 25 30 cgc ccc ggc cct gac cgc agc tct cgc gccgtc gcc ctc ctc ggt gta 261 Arg Pro Gly Pro Asp Arg Ser Ser Arg Ala ValAla Leu Leu Gly Val 35 40 45 cga act cat gtc acg gct gtt gat caa gcc attgta aaa ggt gat aca 309 Arg Thr His Val Thr Ala Val Asp Gln Ala Ile ValLys Gly Asp Thr 50 55 60 65 aag ttg gaa ggt cct gtg gtt gtt gtt act ggtgct tcc agg ggg att 357 Lys Leu Glu Gly Pro Val Val Val Val Thr Gly AlaSer Arg Gly Ile 70 75 80 gga aaa gcc act gca ttg gct ctt gga aaa gca ggctgc aag gtc ttg 405 Gly Lys Ala Thr Ala Leu Ala Leu Gly Lys Ala Gly CysLys Val Leu 85 90 95 gtg aat tat gct cga tct tca aag gag gct gaa gaa gtctcc aag gag 453 Val Asn Tyr Ala Arg Ser Ser Lys Glu Ala Glu Glu Val SerLys Glu 100 105 110 att gaa gca tct gga ggc cag gcc att acc ttt gga ggagat gtt tcc 501 Ile Glu Ala Ser Gly Gly Gln Ala Ile Thr Phe Gly Gly AspVal Ser 115 120 125 aaa gag gct gat gtt gaa tct atg ata aaa gtg gct gttgat aca tgg 549 Lys Glu Ala Asp Val Glu Ser Met Ile Lys Val Ala Val AspThr Trp 130 135 140 145 gga acg att gat gta cta gta aat aat gca gga atcaca cgg gac aca 597 Gly Thr Ile Asp Val Leu Val Asn Asn Ala Gly Ile ThrArg Asp Thr 150 155 160 ttg ttg atg aga atg aag aaa tca cag tgg caa gatgcg att gat ttg 645 Leu Leu Met Arg Met Lys Lys Ser Gln Trp Gln Asp AlaIle Asp Leu 165 170 175 aat ctt aca ggc gtt ttc ctt tgc acg cag gct gcaaca aaa gta atg 693 Asn Leu Thr Gly Val Phe Leu Cys Thr Gln Ala Ala ThrLys Val Met 180 185 190 atg aag aag aaa aag gga aga att atc aat ata gcatcg gtt gtt ggt 741 Met Lys Lys Lys Lys Gly Arg Ile Ile Asn Ile Ala SerVal Val Gly 195 200 205 ctt act ggt aat gct gga caa gct aat tat gct gctgcc aag gct ggg 789 Leu Thr Gly Asn Ala Gly Gln Ala Asn Tyr Ala Ala AlaLys Ala Gly 210 215 220 225 gtt att ggg ttc aca aaa aca gtt gct agg gagtat gcc agc aga aat 837 Val Ile Gly Phe Thr Lys Thr Val Ala Arg Glu TyrAla Ser Arg Asn 230 235 240 att aat gca aac gtt atc gct cct gga ttt attgct tca gat atg act 885 Ile Asn Ala Asn Val Ile Ala Pro Gly Phe Ile AlaSer Asp Met Thr 245 250 255 gct gaa ctt ggt gaa gag tta gag aag aaa attctg tca act att cct 933 Ala Glu Leu Gly Glu Glu Leu Glu Lys Lys Ile LeuSer Thr Ile Pro 260 265 270 tta ggg cgc tat ggt cgg cca gag gat gta gcaggc ctg gtg gaa ttc 981 Leu Gly Arg Tyr Gly Arg Pro Glu Asp Val Ala GlyLeu Val Glu Phe 275 280 285 tta gcc ctc agc cct gct gca agc tac atc actgga cag gtc ctc acc 1029 Leu Ala Leu Ser Pro Ala Ala Ser Tyr Ile Thr GlyGln Val Leu Thr 290 295 300 305 atc gat gga gga atg gta atg taaggcttcgaatctgtgcc gctggcctct 1080 Ile Asp Gly Gly Met Val Met 310 aatgtgtcgcagaaaaaaaa tgtaattcag ttttttgagt gtcattttta aggggtggtt 1140 tcttttgtccgcagcggttt gtggtatagt acagtttgtt tcgaagggag agttgatact 1200 agaaatttgcacacgtatag ttagcttaat ttctttgcga ttggccgatt gctccaaaaa 1260 aaaaaaaaaaaaaaaaaaaa aaaaaa 1286 4 312 PRT Zea mays 4 Met Ala Ala Ala Thr Ala AlaAla Ala Ala Leu Ala Ser Pro Ala Gly 1 5 10 15 Leu Ser Thr Ser Leu AlaArg Arg Gly Leu Val Ser Phe Ala Pro Ala 20 25 30 Leu Arg Pro Gly Pro AspArg Ser Ser Arg Ala Val Ala Leu Leu Gly 35 40 45 Val Arg Thr His Val ThrAla Val Asp Gln Ala Ile Val Lys Gly Asp 50 55 60 Thr Lys Leu Glu Gly ProVal Val Val Val Thr Gly Ala Ser Arg Gly 65 70 75 80 Ile Gly Lys Ala ThrAla Leu Ala Leu Gly Lys Ala Gly Cys Lys Val 85 90 95 Leu Val Asn Tyr AlaArg Ser Ser Lys Glu Ala Glu Glu Val Ser Lys 100 105 110 Glu Ile Glu AlaSer Gly Gly Gln Ala Ile Thr Phe Gly Gly Asp Val 115 120 125 Ser Lys GluAla Asp Val Glu Ser Met Ile Lys Val Ala Val Asp Thr 130 135 140 Trp GlyThr Ile Asp Val Leu Val Asn Asn Ala Gly Ile Thr Arg Asp 145 150 155 160Thr Leu Leu Met Arg Met Lys Lys Ser Gln Trp Gln Asp Ala Ile Asp 165 170175 Leu Asn Leu Thr Gly Val Phe Leu Cys Thr Gln Ala Ala Thr Lys Val 180185 190 Met Met Lys Lys Lys Lys Gly Arg Ile Ile Asn Ile Ala Ser Val Val195 200 205 Gly Leu Thr Gly Asn Ala Gly Gln Ala Asn Tyr Ala Ala Ala LysAla 210 215 220 Gly Val Ile Gly Phe Thr Lys Thr Val Ala Arg Glu Tyr AlaSer Arg 225 230 235 240 Asn Ile Asn Ala Asn Val Ile Ala Pro Gly Phe IleAla Ser Asp Met 245 250 255 Thr Ala Glu Leu Gly Glu Glu Leu Glu Lys LysIle Leu Ser Thr Ile 260 265 270 Pro Leu Gly Arg Tyr Gly Arg Pro Glu AspVal Ala Gly Leu Val Glu 275 280 285 Phe Leu Ala Leu Ser Pro Ala Ala SerTyr Ile Thr Gly Gln Val Leu 290 295 300 Thr Ile Asp Gly Gly Met Val Met305 310 5 1398 DNA Glycine max CDS (122)...(1081) 5 cccaaagcactaatctaaca aacgcaattt aaaaaccgca acaaactttc tctcttgcgt 60 tcacacttattctctggctt cttcttccat tttcgcttct cgtagcgttt tcggaacagt 120 t atg gct tccatt gcc gga tcc aac tgc gtc gct ctc cga acc gcc aac 169 Met Ala Ser IleAla Gly Ser Asn Cys Val Ala Leu Arg Thr Ala Asn 1 5 10 15 ttc ggc gcctcc ggt aac cgg aaa atc ggc cag atc cgc caa tgg tct 217 Phe Gly Ala SerGly Asn Arg Lys Ile Gly Gln Ile Arg Gln Trp Ser 20 25 30 ccg att ctc acgaat ctc cgt ccc gtt tcc ggt ctt cgt cac cga tcg 265 Pro Ile Leu Thr AsnLeu Arg Pro Val Ser Gly Leu Arg His Arg Ser 35 40 45 aat act ccg ttt agctcc tcc ggt gtg aga gca cag gtt gct act ctg 313 Asn Thr Pro Phe Ser SerSer Gly Val Arg Ala Gln Val Ala Thr Leu 50 55 60 gag gaa gca gga acc ggagca act cag aaa gtg gaa gcg ccg gtt gca 361 Glu Glu Ala Gly Thr Gly AlaThr Gln Lys Val Glu Ala Pro Val Ala 65 70 75 80 gtg gtg acc gga gct tccaga ggc att ggc aaa gcg att gca ctg tca 409 Val Val Thr Gly Ala Ser ArgGly Ile Gly Lys Ala Ile Ala Leu Ser 85 90 95 tta ggt aaa gca ggt tgc aaggtt ctg gtc aac tat gca agg tca tcc 457 Leu Gly Lys Ala Gly Cys Lys ValLeu Val Asn Tyr Ala Arg Ser Ser 100 105 110 aag gaa gct gag gag gtt tccaag gag att gag gag ttt ggt ggt caa 505 Lys Glu Ala Glu Glu Val Ser LysGlu Ile Glu Glu Phe Gly Gly Gln 115 120 125 gct ctt aca ttt ggt gga gatgtt tct aac gag gct gat gtg gag tct 553 Ala Leu Thr Phe Gly Gly Asp ValSer Asn Glu Ala Asp Val Glu Ser 130 135 140 atg att aaa act gca gtt gatgct tgg gga aca gtt gat gta tta ata 601 Met Ile Lys Thr Ala Val Asp AlaTrp Gly Thr Val Asp Val Leu Ile 145 150 155 160 aac aat gca gga ata acaaga gat ggt tta tta atg aga atg aag aaa 649 Asn Asn Ala Gly Ile Thr ArgAsp Gly Leu Leu Met Arg Met Lys Lys 165 170 175 tct caa tgg cag gat gttatt gat cta aat ctc act ggt gtt ttt ctt 697 Ser Gln Trp Gln Asp Val IleAsp Leu Asn Leu Thr Gly Val Phe Leu 180 185 190 tgc aca cag gct gct gctaag att atg atg aag aaa aag aag gga agg 745 Cys Thr Gln Ala Ala Ala LysIle Met Met Lys Lys Lys Lys Gly Arg 195 200 205 atc gtc aat att gca tcagtt gtt ggt ttg gtt ggc aat gtt gga caa 793 Ile Val Asn Ile Ala Ser ValVal Gly Leu Val Gly Asn Val Gly Gln 210 215 220 gcc aat tat agt gct gcaaaa gca gga gta att ggc ctg aca aaa act 841 Ala Asn Tyr Ser Ala Ala LysAla Gly Val Ile Gly Leu Thr Lys Thr 225 230 235 240 gtt gcg aag gaa tatgct agt aga aac atc act gtt aat gca gtt gct 889 Val Ala Lys Glu Tyr AlaSer Arg Asn Ile Thr Val Asn Ala Val Ala 245 250 255 cca ggg ttt att gcatct gac atg act gcc aag cta gga caa gac att 937 Pro Gly Phe Ile Ala SerAsp Met Thr Ala Lys Leu Gly Gln Asp Ile 260 265 270 gag aaa aag att ttggag aca atc cca tta gga aga tat ggc caa cca 985 Glu Lys Lys Ile Leu GluThr Ile Pro Leu Gly Arg Tyr Gly Gln Pro 275 280 285 gag gaa gtt gct ggactg gtt gaa ttc ttg gct ctt aat caa gct gcc 1033 Glu Glu Val Ala Gly LeuVal Glu Phe Leu Ala Leu Asn Gln Ala Ala 290 295 300 agt tac atc act gggcag gtt ttc acc att gat gga ggt atg gtg atg 1081 Ser Tyr Ile Thr Gly GlnVal Phe Thr Ile Asp Gly Gly Met Val Met 305 310 315 320 taaattagcaatccttttac cttgcaacat gagcttttgc acttttaaac tacttctgta 1141 cggtgatagtttgttctttg ctgagttttg tttaagctag ttttaccctg tgatttatcg 1201 tagaatattagttgaaatgc aattcagtca cttttggcac tagctctgaa ttgcttactg 1261 atgaaatgcaatgtgtccag catcttgtac cattttggtt tttatggtca tgtcagcaga 1321 ataggcataattcttatatg caattcagtc acttttggca ctaaaaaaaa aaaaaaaaaa 1381 aaaaaaaaaaaaaaaaa 1398 6 320 PRT Glycine max 6 Met Ala Ser Ile Ala Gly Ser Asn CysVal Ala Leu Arg Thr Ala Asn 1 5 10 15 Phe Gly Ala Ser Gly Asn Arg LysIle Gly Gln Ile Arg Gln Trp Ser 20 25 30 Pro Ile Leu Thr Asn Leu Arg ProVal Ser Gly Leu Arg His Arg Ser 35 40 45 Asn Thr Pro Phe Ser Ser Ser GlyVal Arg Ala Gln Val Ala Thr Leu 50 55 60 Glu Glu Ala Gly Thr Gly Ala ThrGln Lys Val Glu Ala Pro Val Ala 65 70 75 80 Val Val Thr Gly Ala Ser ArgGly Ile Gly Lys Ala Ile Ala Leu Ser 85 90 95 Leu Gly Lys Ala Gly Cys LysVal Leu Val Asn Tyr Ala Arg Ser Ser 100 105 110 Lys Glu Ala Glu Glu ValSer Lys Glu Ile Glu Glu Phe Gly Gly Gln 115 120 125 Ala Leu Thr Phe GlyGly Asp Val Ser Asn Glu Ala Asp Val Glu Ser 130 135 140 Met Ile Lys ThrAla Val Asp Ala Trp Gly Thr Val Asp Val Leu Ile 145 150 155 160 Asn AsnAla Gly Ile Thr Arg Asp Gly Leu Leu Met Arg Met Lys Lys 165 170 175 SerGln Trp Gln Asp Val Ile Asp Leu Asn Leu Thr Gly Val Phe Leu 180 185 190Cys Thr Gln Ala Ala Ala Lys Ile Met Met Lys Lys Lys Lys Gly Arg 195 200205 Ile Val Asn Ile Ala Ser Val Val Gly Leu Val Gly Asn Val Gly Gln 210215 220 Ala Asn Tyr Ser Ala Ala Lys Ala Gly Val Ile Gly Leu Thr Lys Thr225 230 235 240 Val Ala Lys Glu Tyr Ala Ser Arg Asn Ile Thr Val Asn AlaVal Ala 245 250 255 Pro Gly Phe Ile Ala Ser Asp Met Thr Ala Lys Leu GlyGln Asp Ile 260 265 270 Glu Lys Lys Ile Leu Glu Thr Ile Pro Leu Gly ArgTyr Gly Gln Pro 275 280 285 Glu Glu Val Ala Gly Leu Val Glu Phe Leu AlaLeu Asn Gln Ala Ala 290 295 300 Ser Tyr Ile Thr Gly Gln Val Phe Thr IleAsp Gly Gly Met Val Met 305 310 315 320 7 1248 DNA Glycine max CDS(16)...(912) 7 cagaaatcaa gtaag atg ggt tct ctg gcc cga cca aac tca ctcttt ttt 51 Met Gly Ser Leu Ala Arg Pro Asn Ser Leu Phe Phe 1 5 10 cgaacc aaa gga ccc gga cgt gcc cgg aaa gta cca agt cag gtt ttg 99 Arg ThrLys Gly Pro Gly Arg Ala Arg Lys Val Pro Ser Gln Val Leu 15 20 25 gct tttcag cgt tcc aat tca aat ggt tca ttt ccc tca tca gaa cag 147 Ala Phe GlnArg Ser Asn Ser Asn Gly Ser Phe Pro Ser Ser Glu Gln 30 35 40 cta gaa cttgaa gca agc cag aag aac atg gaa gca cct gtt gtt gta 195 Leu Glu Leu GluAla Ser Gln Lys Asn Met Glu Ala Pro Val Val Val 45 50 55 60 gtc act ggagcc tcc aga ggc att ggc cgt gca att gca ctt tcc ttg 243 Val Thr Gly AlaSer Arg Gly Ile Gly Arg Ala Ile Ala Leu Ser Leu 65 70 75 ggt aaa gcc ccatgc aag gtg ttg gtc aac tat gcc agg tca tcc atg 291 Gly Lys Ala Pro CysLys Val Leu Val Asn Tyr Ala Arg Ser Ser Met 80 85 90 caa gct gag gag gtttcc aac ttg att gag gcg ttt ggt gga caa gct 339 Gln Ala Glu Glu Val SerAsn Leu Ile Glu Ala Phe Gly Gly Gln Ala 95 100 105 ctt acc ttc gag ggagat gtt tca aat gag gcc gat gtg gaa tct atg 387 Leu Thr Phe Glu Gly AspVal Ser Asn Glu Ala Asp Val Glu Ser Met 110 115 120 att aga act gca gttgat gct tgg gga act gtt gat gta ttg gta aac 435 Ile Arg Thr Ala Val AspAla Trp Gly Thr Val Asp Val Leu Val Asn 125 130 135 140 aat gca gga attact cga gat ggt ttg tta atg aga atg aag aaa tca 483 Asn Ala Gly Ile ThrArg Asp Gly Leu Leu Met Arg Met Lys Lys Ser 145 150 155 caa tgg cag gaagtt att gat ctg aat ctc act ggt gtt ttt ctt tgc 531 Gln Trp Gln Glu ValIle Asp Leu Asn Leu Thr Gly Val Phe Leu Cys 160 165 170 atg cag gca gcagca aag att atg acg atg aaa aag aag gga agg ata 579 Met Gln Ala Ala AlaLys Ile Met Thr Met Lys Lys Lys Gly Arg Ile 175 180 185 atc aat att acatca gtt att ggt cag gtt ggc aat gtt gga caa gcc 627 Ile Asn Ile Thr SerVal Ile Gly Gln Val Gly Asn Val Gly Gln Ala 190 195 200 aat tat agt gctgca aag gca ggg gta att ggc ctc aca aaa agt gct 675 Asn Tyr Ser Ala AlaLys Ala Gly Val Ile Gly Leu Thr Lys Ser Ala 205 210 215 220 gcc agg gaatat gct agc aga aac atc act gtt aat gca gta gcc cct 723 Ala Arg Glu TyrAla Ser Arg Asn Ile Thr Val Asn Ala Val Ala Pro 225 230 235 ggg ttt attgca tct gat atg act gcc aat cta cga cca ggc att gag 771 Gly Phe Ile AlaSer Asp Met Thr Ala Asn Leu Arg Pro Gly Ile Glu 240 245 250 aaa aaa agattg gaa tta atc ccc tta gga aga ctt ggc caa cca gaa 819 Lys Lys Arg LeuGlu Leu Ile Pro Leu Gly Arg Leu Gly Gln Pro Glu 255 260 265 gaa gtt gctgga ctt gtg gaa ttc ttg gct ctt aat cct gct gcc aat 867 Glu Val Ala GlyLeu Val Glu Phe Leu Ala Leu Asn Pro Ala Ala Asn 270 275 280 tac atc actggg cag gtg ttc acc att gat gga ggt ttg gca atg 912 Tyr Ile Thr Gly GlnVal Phe Thr Ile Asp Gly Gly Leu Ala Met 285 290 295 tgagtctcaggaatctgttt ccgtatatag caacttgaac ttctttactt cacagttcat 972 ctcaaaggccacagaatttc aacttctgtc atggtgctag tttgttctat gctgatttat 1032 gctcaagctagtaatacgtg gtgacttatt gtagaagttt agttgagctt tttaacaggt 1092 tgcttcttgagatgaattcg acatattgct gcattttggt gactcttatg ggtcacatta 1152 cattttacatttcctgcaat ttaccaattt tgggccttat ctttccctaa tgatgagatg 1212 cttgcggtcatttcggaata aaaaaaaaaa aaaaaa 1248 8 299 PRT Glycine max 8 Met Gly SerLeu Ala Arg Pro Asn Ser Leu Phe Phe Arg Thr Lys Gly 1 5 10 15 Pro GlyArg Ala Arg Lys Val Pro Ser Gln Val Leu Ala Phe Gln Arg 20 25 30 Ser AsnSer Asn Gly Ser Phe Pro Ser Ser Glu Gln Leu Glu Leu Glu 35 40 45 Ala SerGln Lys Asn Met Glu Ala Pro Val Val Val Val Thr Gly Ala 50 55 60 Ser ArgGly Ile Gly Arg Ala Ile Ala Leu Ser Leu Gly Lys Ala Pro 65 70 75 80 CysLys Val Leu Val Asn Tyr Ala Arg Ser Ser Met Gln Ala Glu Glu 85 90 95 ValSer Asn Leu Ile Glu Ala Phe Gly Gly Gln Ala Leu Thr Phe Glu 100 105 110Gly Asp Val Ser Asn Glu Ala Asp Val Glu Ser Met Ile Arg Thr Ala 115 120125 Val Asp Ala Trp Gly Thr Val Asp Val Leu Val Asn Asn Ala Gly Ile 130135 140 Thr Arg Asp Gly Leu Leu Met Arg Met Lys Lys Ser Gln Trp Gln Glu145 150 155 160 Val Ile Asp Leu Asn Leu Thr Gly Val Phe Leu Cys Met GlnAla Ala 165 170 175 Ala Lys Ile Met Thr Met Lys Lys Lys Gly Arg Ile IleAsn Ile Thr 180 185 190 Ser Val Ile Gly Gln Val Gly Asn Val Gly Gln AlaAsn Tyr Ser Ala 195 200 205 Ala Lys Ala Gly Val Ile Gly Leu Thr Lys SerAla Ala Arg Glu Tyr 210 215 220 Ala Ser Arg Asn Ile Thr Val Asn Ala ValAla Pro Gly Phe Ile Ala 225 230 235 240 Ser Asp Met Thr Ala Asn Leu ArgPro Gly Ile Glu Lys Lys Arg Leu 245 250 255 Glu Leu Ile Pro Leu Gly ArgLeu Gly Gln Pro Glu Glu Val Ala Gly 260 265 270 Leu Val Glu Phe Leu AlaLeu Asn Pro Ala Ala Asn Tyr Ile Thr Gly 275 280 285 Gln Val Phe Thr IleAsp Gly Gly Leu Ala Met 290 295 9 1680 DNA Pseudomonas fluorescensmisc_feature (1)...(1680) SEQ ID NO1 from WO 01/23580 9 atgagcaacaagaacaatga agacctgcag cgccaagcct ctgagaatac cctcgggctg 60 aacccggtgatcggcatccg cggcaaggat ctgctgacct ccgcgcgcat ggtcatgctg 120 caggccatcaagcagccctt ccacagtgcc aagcacgtcg cccatttcgg ggtcgagctt 180 aaaaacgtcctgctcggctc ttcggccctg cagccggaag ccgacgaccg tcgcttcgcg 240 gacccggcctggagccagaa ccccctctac aagcgctacc tgcagaccta cctcgcctgg 300 cgcaaggaactgcaccagtg gatcgagcac agcgacctgt cgtcgtccga taccagccgc 360 ggccacttcgtgatcaacct gatgaccgaa gccatggccc ccaccaacac catggccaac 420 ccggcggcggtgaagcgctt cttcgaaacc ggcggcaaga gcctgctcga cggcctctcg 480 cacctggccaaggacctggt caacaacggc ggcatgccca gccaggtcaa catggacgcc 540 ttcgaggtcggcaagaacct cgccaccacc gaaggcgccg tggtcttccg caatgacgtg 600 ctggagctgatccagtacaa gcccatcacc gagcaggtgc acgagcgccc gctgctggtg 660 gtgccgccgcagatcaacaa gttctacgtc ttcgacctgt cccaggagaa gagcctggcg 720 cgcttcaacctgcgcaacgg catccagacc ttcatcgtca gctggcgcaa cccgaccaag 780 gcccagcgcgaatggggcct gtcgacctac atcgaggcgc tcaaggaaac catcgaggtg 840 gtgctgaagatcaccggcgc caaggacctc aacatgctcg gtgcctgctc cggcggcatc 900 accacggtcgccctgctggg ccactaccag gcgatcggcg agcacaaggt gaacgccttc 960 acgcagttggtcagcgtgct cgacttcaac ctggacaccc aggtcgcgct gttcgccgac 1020 gaaaccaccctggaggccgc caagcgccgc tcctaccagt ccggcgtgct ggaaggcaag 1080 gaaatggccaaggtcttcgc ctggatgcgc cccaacgacc tgatctggaa ctactgggtg 1140 aacaactacctgctcggcaa cgagccgccg gtgttcgaca tcctctactg gaacaacgac 1200 accacgcgcctgcccgccgc cttccacggc gagttggtgg agatgttcaa gaccaacccg 1260 ctgacccgccccgacgggct ggaggtctgc ggcaccccaa tcgacctaaa gaaggtcacc 1320 tgcgacttcttctgcgtggc cggcaccacc gaccacatca ccccttggga agcctgctac 1380 cgctccgcccgcctgctggg cggcaaatgc gagttcgtgc tgtccaacag cgggcacatc 1440 cagagcatcctcaacccccc gggcaacccc aaggcgcgct tctccaccaa cagcgagatg 1500 ccggcggacccgaaggagtg gcaggaaaac gccaccaagc acgccgactc ctggtggctg 1560 tactggcaaacctggctggc ggagcgctcg ggcaagacca agaaagccag cttcaccctc 1620 ggcaacaaggcctacccggc cggcgaggct tcgccaggga cctatgtcca cgaacgttga 1680 10 1683 DNAPseudomonas fluorescens misc_feature (1)...(1683) SEQ ID NO3 from WO01/23580 10 atgcgagaga aacaggtgtc gggagccttg ccggtccccg ctaactacatgaacgcgcag 60 agcgccattg tcggcttgcg aggcaaggac ctggcctcca ccgtccgcaccctcgccctg 120 cagggcttga agcaccccgt gcacagcgcc cgccacgtcc tcgccttcggcggccagctg 180 ggccgcgtat tgatgggcga caccccgcac aagcccaacc cgcaggacgcgcgcttcgcc 240 gatccctcct ggagccacaa cccgttctac cgtcgcggct tgcaggcctacctggcctgg 300 cagaaacaac tctatgcctg ggtcgaggac agcgacctca ccgacgatgaccgcgcccgt 360 gcgcgcttcg tcctggccct ggtcagcgac gccatggcgc cctccaacagcctgctcaac 420 cccctcgcgg tgaaggagct gttcaacacc ggcggcctca gcctgctcaatggcgcgcgc 480 cacctgctgg acgatgtgct gaacaacaac gccatgccgc gccaggtcagcaagcactcc 540 ttcgagatcg gccgcaacct ggcaaccacg cccgggtcgg tggtctatcgcaacgagctg 600 ctggaactga tccagtacaa gccgatgagc gagaagcagt acctcaagcctctgctgatc 660 gtcccgccgc aaatcaacaa gttctacatc ttcgacctct cgccggagaagagcttcgtc 720 cagtacgcgc tgaagaatgg cctgcaggtg ctcatggtca gctggcgcaaccccgatgcg 780 cgccaccgcg aatggggcct gtccacctat gtgcaggcgc tggagcaggcggtcgacgtg 840 gcccgcgcca tcaccggcag caaggacgtc aacctgatgg gcgcctgcgccggcggcctg 900 accatcgccg ccctgcaggg ccacctccag gccaagcgcc aactacgcaaggtcagcagc 960 gccagctacc tggtcagcct gctggacagc caggtcgaaa gccccgccgccctgttcgcc 1020 gacgaacaga ccctggaggc ggccaagcgc cgctcctacc agcacggcgtcctggacggc 1080 cgcgacatgg cgaagatctt cgcctggatg cgccccaacg acctggtgtggaactacttc 1140 gtcaacaact acctgctggg ccgtcagccg ccggccttcg acatcctctactggaacaac 1200 gacaacaccc gcctgcccgc cgccttccac ggcgacctgc tggacttcttcaagcacaac 1260 ccgctgaccc ggggcggcgc gctggaaatc tgcggcaccc ccatcgacctgcagaaggtc 1320 acggtggaca gcttcagcgt ggccggtatc aacgaccaca tcaccccctgggacgcggtc 1380 tatcgctcgg cgcggctgct gggtggcgag agccgcttcg tgctgtccaacagcgggcac 1440 atccagagca tcctcaaccc accgggcaac cccaaggcca actacctggaaaacggcaag 1500 ctcagctcgg accaccgcgc ctggtactac gacgcgaaga acgtgcagggcagctggtgg 1560 ccggagtggc tgagctggat ccaggcgcgc tcgggggagc agcgcgaaaccctggtcacc 1620 ctcggcaacc aggcccaccc acccatggag gcggcacccg gcacctacgtgcacgtgcgc 1680 tga 1683

That which is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a maize 3-oxoacyl-[ACP] reductase (OAR);(b) a nucleotide sequence encoding a soybean OAR; (c) a nucleotidesequence comprising the nucleotide sequence set forth in SEQ ID NO: 1,3, 5, or 7; (d) a nucleotide sequence which encodes a polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 8; (e) anucleotide sequence comprising at least 15 contiguous bases of at leastone nucleotide sequence selected from the group consisting of thenucleotide sequences set forth in SEQ ID NO: 1, 3, 5, and 7, whereinsaid nucleotide sequence encodes a polypeptide comprising OAR activity;(f) a nucleotide sequence having at least 85% sequence identity to atleast one nucleotide sequence selected from the group consisting of thenucleotide sequences set forth in SEQ ID NO: 1, 3, 5, and 7, whereinsaid nucleotide sequence encodes a polypeptide comprising OAR activity;(g) a nucleotide sequence comprising at least 20 nucleotides in lengthwhich hybridizes under low stringency conditions to at least onenucleotide sequence selected from the group consisting of the nucleotidesequences set forth in SEQ ID NO: 1, 3, 5, and 7, wherein saidnucleotide sequence encodes a polypeptide comprising OAR activity; and(h) a nucleotide sequence complementary to a nucleotide sequence of (a),(b), (c), (d), (e), (f), or (g), wherein said nucleotide sequence iscapable of antisense suppression of OAR expression in a cell.
 2. Anexpression cassette comprising at least one nucleic acid molecule ofclaim 1 operably linked to a promoter.
 3. The expression cassette ofclaim 2, wherein said promoter drives expression in a plant cell.
 4. Theexpression cassette of claim 2, wherein said nucleic acid molecule isoperably linked in an antisense orientation to said promoter.
 5. Theexpression cassette of claim 3 further comprising a peroxisome-targetingsignal operably linked to said nucleic acid molecule.
 6. A non-humanhost cell transformed with at least one expression cassette of claim 2.7. The host cell of claim 6, wherein said host cell is selected from thegroup consisting of a plant cell, a bacterial cell, and a yeast cell. 8.A transgenic plant comprising in its genome a stably integrated firstnucleotide construct comprising a nucleic acid molecule operably linkedto a first promoter that drives expression in a plant cell, wherein saidnucleic acid molecule comprises a first nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding a maizeOAR; (b) a nucleotide sequence encoding a soybean OAR; (c) a nucleotidesequence comprising the nucleotide sequence set forth in SEQ ID NO: 1,3, 5, or 7; (d) a nucleotide sequence which encodes a polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 8; (e) anucleotide sequence comprising at least 15 contiguous bases of at leastone nucleotide sequence selected from the group consisting of thenucleotide sequences set forth in SEQ ID NO: 1, 3, 5, and 7, whereinsaid nucleotide sequence encodes a polypeptide comprising OAR activity;(f) a nucleotide sequence having at least 85% sequence identity to atleast one nucleotide sequence selected from the group consisting of thenucleotide sequences set forth in SEQ ID NO: 1, 3, 5, and 7, whereinsaid nucleotide sequence encodes a polypeptide comprising OAR activity;(g) a nucleotide sequence comprising at least 20 nucleotides in lengthwhich hybridizes under low stringency conditions to at least onenucleotide sequence selected from the group consisting of the nucleotidesequences set forth in SEQ ID NO: 1, 3, 5, and 7, wherein saidnucleotide sequence encodes a polypeptide comprising OAR activity; and(h) a nucleotide sequence complementary to a nucleotide sequence of (a),(b), (c), (d), (e), (f), or (g), wherein said nucleotide sequence iscapable of antisense suppression of OAR expression in a cell.
 9. Theplant of claim 8, wherein said plant is a monocot or a dicot.
 10. Theplant of claim 9, wherein said plant is selected from the groupconsisting of corn, soybean, wheat, rice, alfalfa, barley, millet,sunflower, sorghum, safflower, Brassica spp., and cotton. 11.Transformed seed of the plant of claim
 8. 12. The plant of claim 8further comprising in its genome a stably integrated second nucleotideconstruct comprising a second promoter that drives expression in a plantcell operably linked to a second nucleotide sequence, wherein saidsecond nucleotide sequence encodes a polyhydroxyalkanoate (PHA) synthase13. The plant of claim 12, wherein said PHA synthase is encoded by anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a bacterial PHA synthase; and (b) anucleotide sequence selected from the group consisting of the nucleotidesequences set forth in SEQ ID NO:9, SEQ ID NO:10, GenBank Accession No.M58445, GenBank Accession No. AF042276, EMBL Accession No. A49465, EMBLAccession No. X66592, and DDBJ Accession No. D88825.
 14. The plant ofclaim 13, wherein said PHA synthase is capable of utilizing C₄-C₁₈ assubstrate.
 15. The plant of claim 13, wherein said substrate isD-3-hydroxyacyl-CoA.
 16. The plant of claim 13, further comprising inits genome a stably integrated third nucleotide construct comprising athird promoter that drives expression in a plant cell operably linked toa third nucleotide sequence, wherein said third nucleotide sequenceencodes a PHA synthase capable of synthesizing polyhydroxybutyrate. 17.The plant of claim 16, wherein said third nucleotide sequence comprisesa nucleotide sequence selected from the group consisting of: (a) anucleotide sequence set forth in GenBank Accession No. J05003; (b) anucleotide sequence set forth in GenBank Accession No. U04848; (c) anucleotide sequence set forth in GenBank Accession No. AF078795; (d) anucleotide sequence set forth in EMBL Accession No. AJ006237; (e) anucleotide sequence set forth in DDBJ Accession No. AB009237; (g) anucleotide sequence set forth in GenBank Accession No. L07893; (h) anucleotide sequence set forth in DDBJ Accession No. D43764; and (i) anucleotide sequence set forth in GenBank Accession No. U66242.
 18. Theplant of claim 16, wherein at least one of said first, second, and thirdnucleotide sequences is operably linked to a nucleotide sequenceencoding a peroxisome-targeting signal.
 19. The plant of claim 16,wherein at least one of said first, second, and third promoters isselected from the group consisting of seed-preferred promoters,chemical-regulatable promoters, germination-preferred promoters, andleaf-preferred promoters.
 20. The plant of claim 16, wherein said plantproduces PHA in at least one cellular compartment in seeds selected fromthe group consisting of the cytosol, the plastids, and the peroxisomes21. A method for producing PHA copolymers in a plant, comprising: (a)providing a plant of claim 16; (b) growing said plant under conditionsfavorable for the synthesis of said PHA copolymers in said plant; (c)allowing sufficient time for said plant to produce said PHA copolymers;(d) harvesting said plant or part thereof containing said PHAcopolymers; and (e) extracting said PHA copolymers from said plant orpart thereof.
 22. An isolated polypeptide comprising a member selectedfrom the group consisting of: (a) a maize OAR; (b) a soybean OAR; (c) apolypeptide comprising the amino acid sequence of SEQ ID NO: 2,4,6, or8; (d) a polypeptide comprising at least 15 contiguous amino acids of atleast one amino acid sequence selected from the group consisting of theamino acid sequences set forth in SEQ ID NOs: 2, 4, 6, and 8, whereinsaid polypeptide comprises OAR activity; (e) a polypeptide havinggreater than 85% sequence identity to at least one amino acid sequenceselected from the group consisting of the amino acid sequences set forthin SEQ ID NOs: 2, 4, 6, and 8, wherein said polypeptide comprises OARactivity; and (f) a polypeptide encoded by the nucleotide sequence setforth in SEQ ID NO: 1, 3, 5, or 7.